ABCMdb

ABCC7 p.Thr338Cys

ClinVar:	c.1012A>G , p.Thr338Ala ? , not provided c.1013C>T , p.Thr338Ile D , Pathogenic
CF databases:	c.1013C>T , p.Thr338Ile D , CF-causing ; CFTR1: A nucleotide change C->T at position 1145 which causes the replacement of a Threonine by Isoleucine residue in codon 338 of exon 7. c.1012A>G , p.Thr338Ala (CFTR1) ? , This mutation was identified in one Iranian CBAVD patient.
Predicted by SNAP2:	A: D (85%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (53%), K: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (91%), V: D (85%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: N, Q: D, R: D, S: N, V: N, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] CFTR: what's it like inside the pore? J Exp Zool A Comp Exp Biol. 2003 Nov 1;300(1):69-75. Liu X, Smith SS, Dawson DC
CFTR: what's it like inside the pore?
J Exp Zool A Comp Exp Biol. 2003 Nov 1;300(1):69-75., 2003-11-01 [PMID:14598388]

Abstract [show]
The Cystic Fibrosis Conductance Regulator (CFTR) functions as a cAMP-activated, anion-selective channel, but the structural basis for anion permeation is not well understood. Here we summarize recent studies aimed at understanding how anions move through the CFTR channel, and the nature of the environment anions experience inside the pore. From these studies it is apparent that anion permeability selectivity and anion binding selectivity of the pore are consistent with a model based on a "dielectric tunnel." The selectivity pattern for halides and pseudohalides can be predicted if it is assumed that permeant anions partition between bulk water and a polarizable space that is characterized by an effective dielectric constant of about 19. Covalent labeling of engineered cysteines and pH titration of engineered cysteines and histidines lead to the conclusion that the CFTR anion conduction path includes a positively charged outer vestibule. A residue in transmembrane segment 6 (TM6) (R334) appears to reside in the outer vestibule of the CFTR pore where it creates a positive electrostatic potential that enhances anion conduction.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
114 T338C CFTR undergoes pH-dependent changes in gCl and I-V shape that are not seen in wild type, T338A or T338S CFTR. Login to comment
115 Preliminary data indicate that the pH induced changes in T338C CFTR conductance are due to a change in the single channel conductance without a concurrent change in the open probability. Login to comment
117 Titration of the macroscopic conductance due to T338C and T338H CFTR indicates that positive charges at R334, perhaps at K335, and perhaps elsewhere may cause the pKa of T338C CFTR to become more acidic than in free solution (E7.4) and to render T338H CFTR non-titratable. Login to comment
118 Changing the charge at position 334 either by modification of R334C/T338H CFTR with polar thiol reactive reagents or by amino acid substitution (R334A/T338C) shifts the titration curve in a direction that was predicted on the basis of a nearby positive charge being able to stabilize a titratable group (Liu et al., 2001). Login to comment
202 CFTR: pH titration and chemical modification indicate that T338C (TM6) lies on the outward-facing, water-accessible surface of the protein. Login to comment

[hide] Variable reactivity of an engineered cysteine at p... J Biol Chem. 2006 Mar 24;281(12):8275-85. Epub 2006 Jan 24. Liu X, Alexander C, Serrano J, Borg E, Dawson DC
Variable reactivity of an engineered cysteine at position 338 in cystic fibrosis transmembrane conductance regulator reflects different chemical states of the thiol.
J Biol Chem. 2006 Mar 24;281(12):8275-85. Epub 2006 Jan 24., 2006-03-24 [PMID:16436375]

Abstract [show]
In a previous study of T338C CFTR (cystic fibrosis transmembrane conductance regulator) we found that protons and thiol-directed reagents modified channel properties in a manner consistent with the hypothesis that this residue lies within the conduction path, but the observed reactivity was not consistent with the presence of a single thiolate species in the pore. Here we report results consistent with the notion that the thiol moiety can exist in at least three chemical states, the simple thiol, and two altered states. One of the altered states displays reactivity toward thiols like dithiothreitol and 2-mercaptoethanol as well as reagents: mixed disulfides (methanethiosulfonate reagents: MTSET+, MTSES-) and an alkylating agent (iodoacetamide). The other altered state is unreactive. The phenotype associated with the reactive, altered state could be replicated by exposing oocytes expressing T338C CFTR to CuCl2, but not by glutathionylation or nitrosylation of the thiol or by oxidation with hydrogen peroxide. The results are consistent with the hypothesis that substituting a cysteine at 338 can create an adventitious metal binding site. Metal liganding alters thiol reactivity and may, in some cases, catalyze oxidation of the thiol to an unreactive form such as a sulfinic or sulfonic acid.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
0 Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol* Received for publication,November 21, 2005, and in revised form, January 23, 2006 Published, JBC Papers in Press,January 24, 2006, DOI 10.1074/jbc.M512458200 Xuehong Liu1 , Christopher Alexander, Jose Serrano2 , Erik Borg, and David C. Dawson3 From the Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239 In a previous study of T338C CFTR (cystic fibrosis transmembrane conductance regulator) we found that protons and thiol-directed reagents modified channel properties in a manner consistent with the hypothesis that this residue lies within the conduction path, but the observed reactivity was not consistent with the presence of a single thiolate species in the pore. Login to comment
4 The phenotype associated with the reactive, altered state could be replicated by exposing oocytes expressing T338C CFTR to CuCl2, but not by glutathionylation or nitrosylation of the thiol or by oxidation with hydrogen peroxide. Login to comment
21 The T338C CFTR mutants used in this study were generated on two different CFTR backgrounds, either the conventional WT background or a Cys-less background in which all the 18 endogenous cysteines were substituted with alternative amino acids (serine or leucine). Login to comment
25 To distinguish the two T338C mutants, the construct generated on the Cys-less background is labeled as T338C/Cys-less CFTR and that generated on the WT background is labeled T338C or T338C/WT CFTR. Login to comment
53 RESULTS T338C/WT CFTR Conductance Was Markedly Altered by 2-ME or DTT Prior to Exposure to Exogenous Thiol-directed Reagents5 - Exposing oocytes expressing T338C/WT CFTR to 2-ME or DTT during steady state activation led to increases in conductance (without any discernable change in reversal potential) that were rapid (t1/2 ϭ 20 s), and of variable amplitude and were not seen in oocytes expressing CFTR constructs lacking the cysteine at 338, such as WT, T338A, T338H, T338S CFTR, or Cys-less CFTR. Login to comment
54 Shown in Fig. 1A is a representative experiment in which exposing an oocyte expressing T338C/WT CFTR 5 We use the term "thiol-directed reagents" to refer to species like MTS reagents or IAM that react with the thiolate anion to form, for example, a mixed disulfide or an alky- latedthiol.2-MEandDTT(reduced),althoughtheyarewidelyusedinconnectionwith disulfide chemistry, are not, strictly speaking, thiol-directed reagents as they do not react with the thiolate anion. Login to comment
56 A, effect of 2-ME on T338C/WT CFTR conductance. Login to comment
57 Following activation by 10 ␮M Isop and 1 mM isobutylmethylxanthine (hatched bar), an oocyte expressing T338C CFTR was exposed to 1 mM 2-ME (open circles). Login to comment
62 C, effect of 2-ME (open circles) on T338C/ Cys-less CFTR conductance. Inset C1, fractional change in conductance (n ϭ 34) induced by 2-ME orDTTversusinitialconductance.InsetC2,I-Vplots as above. Login to comment
64 Different Chemical States of an Engineered Cysteine (T338C) 8276 to 1 mM 2-ME at steady state activation induced a rapid, over 2-fold increase in conductance (gCl at Vm í Erev). Login to comment
69 Reducing agents had no effect on the conductance of oocytes expressing Cys-less CFTR (Fig. 1B).6 However, in oocytes expressing T338C/Cys-less CFTR, in which a cysteine was substituted at position 338 in the Cys-less background, 1 mM 2-ME induced a variable increase in conductance similar to that seen in oocytes expressing T338C/WT CFTR (Fig. 1C). Login to comment
70 These observations indicated that the cysteine substituted at position 338 was necessary and sufficient to account for the effects of reducing agents seen in oocytes expressing T338C/WT CFTR and ruled out the breaking of an intramolecular disulfide bond within a CFTR monomer as a mechanism for the increase in conductance. Login to comment
73 Furthermore, detailed studies of T338C/WT CFTR suggest that residue 338 lies within the pore (25) where it exhibits reactivity toward mixed disulfides and alkylating agents that is not consistent with a disulfide bond (see also below). Login to comment
75 The response of individual oocytes expressing T338C/WT CFTR to 2-ME or DTT varied widely, as indicated by the results compiled in Fig. 1, inset A1. Login to comment
77 A similar pattern of reactivity was observed in oocytes expressing T338C/Cys-less CFTR (Fig. 1C, inset). Login to comment
83 Evidence for a Terminal Oxidation State-In a previous study of T338C CFTR (25) it became apparent that there was a high degree of variability in the response to thiol-directed reagents even if oocytes had been previously exposed to 2-ME or DTT. Login to comment
85 This phenomenon is explored in more detail in the current study, and the results obtained using a variety of thiol-directed reagents uniformly suggest that whereas a variable subpopulation of T338C channels display altered reactivity that can be rescued by exposure to 2-ME or DTT, there is a second subpopulation of channels that is simply unreactive. Login to comment
86 Oocytes Expressing T338C/WT CFTR Displayed Highly Variable Reactivity Toward MTSETϩ and MTSES- -The disparate response of oocytes expressing T338C CFTRs to 2-ME or DTT is compatible with the notion that, in any individual oocyte, a variable percentage of channels is in the simple thiol form (S- , SH). Login to comment
89 Shown in Fig. 2, A and B, are representative experiments in which, following activation of T338C/WT CFTR, oocytes not previously exposed to 2-ME and DTT were exposed to 1 mM MTSETϩ . Login to comment
103 Different Chemical States of an Engineered Cysteine (T338C) MARCH 24, 2006•VOLUME 281•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8277 could arise because the population of channels that are sensitive to DTT and 2-ME is heterogeneous in some way not discerned in these studies. Login to comment
104 The data summarized by the gray triangles in Fig. 2C demonstrates that exposure of different oocytes expressing T338C/WT CFTR to MTSETϩ under identical conditions, prior to any exposure to 2-ME and DTT, could result in an increase, a decrease, or no change in conductance! Login to comment
110 The observation that reactivity remains highly variable even after exposure to 10-20 mM 2-ME or DTT suggests that a variable population of the T338C channels is simply unreactive, due perhaps to some higher order oxidation of the thiol (32-35). Login to comment
111 It should be noted that the cysteine at position 338 is essential for the effects of MTS reagents as well as 2-ME and DTT shown above, because neither the conductance due to T338A or T338S CFTR was sensitive to reducing agents or thiol-directed reagents.7 Trapping Thiols with an Alkylating Agent, IAM-The results presented so far are compatible with a scheme in which the total conductance of an oocyte expressing T338C/WT CFTR or T338C/Cys-less CFTR comprises at least three components that we will label as gSH, gSX1, and gSX2, where the total conductance, gCl, is given by Equation 1. gCl ϭ gSH ϩ gSX1 ϩ gSX2 (Eq. 1) 7 X. Liu and D. C. Dawson, unpublished observation. Login to comment
114 A, an oocyte expressing T338C/WT CFTR was exposed to: 1 mM MTSETϩ (gray triangles), 5 mM 2-ME (open circles), and 1 mM MTSETϩ . Login to comment
118 C, changes in conductance induced by MTSETϩ or MTSES- (black circles) versus initial conductance of naı¨ve oocytes expressing T338C CFTR. Login to comment
121 Different Chemical States of an Engineered Cysteine (T338C) 8278 Here gSH represents channels in which the cysteine at 338 is in the simple thiol form (S- , SH), and gSX1 and gSX2 represent channels containing one of two altered forms of the cysteine, one that reverts to the simple thiol in the presence of 2-ME and DTT (gSX1) and another (gSX2) that does not. Login to comment
142 Different Chemical States of an Engineered Cysteine (T338C) MARCH 24, 2006•VOLUME 281•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8279 had little or no effect on the macroscopic conductance of pretreated oocytes (Fig. 3B, n ϭ 6). Login to comment
152 Fig. 4B contains the result of an experiment in which alkylation was monitored at pH 9 using a naı¨ve oocyte expressing T338C/WT CFTR (n ϭ 5). Login to comment
164 Different Chemical States of an Engineered Cysteine (T338C) 8280 with the hypothesis that channels reacting with IAM originated from two populations. Login to comment
170 Can Thiol-reactive Agents Mimic Aspects of the Spontaneous Alteration of T338C CFTR Reactivity?-The signature behavior of spontaneously occurring, reversibly altered T338C CFTR channels (gSX1) is a rapid increase in conductance upon exposure to 1 mM 2-ME or DTT (t1/2 Ͻ 20-40 s), a net increase in macroscopic conductance when reacted with MTSETϩ , and relatively slow trapping by IAM. Login to comment
183 T338C CFTR Channels Reacted with Either Glutathione or MTSETϩ Could Not Be Trapped by IAM-Fig. 6A illustrates a typical result of glutathionylation of T338C CFTR channels (n ϭ 3). Login to comment
191 This result is consistent with the expectation that neither mixed disulfide would react with the alkylating agents and strongly suggests that the gSX1 state of the T338C channel is not due to the formation of a mixed disulfide. Login to comment
192 Diamide-GSH had no discernable effect on conductance of oocytes expressing T338A CFTR.7 Oxidation by NO or H2O2 Did Not Reproduce the Signature Behavior of Spontaneously Oxidized T338C CFTR Channels-Fig. 7A depicts a typical experiment (n ϭ 4) in which an oocyte expressing T338C CFTR was first exposed to 1 mM DTT to increase the number of cysteines in the simple thiol form. Exposure to 1 mM SNAP, a commonly used NO donor (46, 65), produced a minimal effect on the conductance, but largely blocked the subsequent reaction with MTSES- , indicating oxida- tionofthecysteinetothenitrosothiol.Thisapparentoxidationwaswithout effect on the macroscopic conductance but was readily reversed by exposing oocytes to 1 mM DTT, as indicated by an 80% decrease in conductance followingthesecondexposuretoMTSES- .SNAPhadnodiscernableeffect on conductance of oocytes expressing T338A CFTR.7 Fig. 7B depicts a typical experiment (n ϭ 2) in which an oocyte expressing T338C CFTR was first exposed to 1 mM DTT to increase the number of cysteines in the simple thiol form. Exposure to 5 mM H2O2 FIGURE 5. Login to comment
197 Different Chemical States of an Engineered Cysteine (T338C) MARCH 24, 2006•VOLUME 281•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8281 produced a decrease in conductance that was not spontaneously reversible. Login to comment
206 Neither SNAP nor H2O2 mimicked the phenotype of T338C CFTR. Login to comment
210 Different Chemical States of an Engineered Cysteine (T338C) 8282 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 12•MARCH 24, 2006 atUniversityofNorthCarolinaatChapelHill,onAugust,2011www.jbc.orgDownloadedfrom pus oocytes (66), so we explored the consequences of exposing oocytes expressing T338C CFTR to metals added to the perfusion solution. Login to comment
213 We have found that many constructs with a single added cysteine exhibit reversible block by Zn2ϩ with K1/2 ranging from 15 ␮M to 1 mM (71, 72).7 In contrast, exposure to low concentrations of copper8 in the perfusate induced a substantial decrease in T338C CFTR conductance that demonstrated a variable but substantial component that was not reversed by washing, indicative of a high affinity interaction. Login to comment
214 At 1 ␮M, copper induced an 80% (Ϯ5%, n ϭ 5) decrease in T338C CFTR conductance, but was without effect on T338A or WT CFTR conductance.9 Washing often produced a slow recovery from inhibition that could vary from near zero to about 32% of the inhibited conductance. Login to comment
217 Exposure of copper-inhibited oocytes expressing T338C/WT to MTSETϩ mimicked the phenotype seen in naı¨ve oocytes. Login to comment
228 Copper mimicked the phenotype seen in naı¨ve oocytes expressing T338C CFTR. Login to comment
233 Different Chemical States of an Engineered Cysteine (T338C) MARCH 24, 2006•VOLUME 281•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8283 reduces macroscopic conductance and alters, but does not eliminate, the reactivity of the thiol toward mixed disulfides and alkylating agents. Login to comment
234 DISCUSSION The Chemical State of a Cysteine Substituted at 338 Can Vary-The results presented here support the hypothesis that when CFTR channels containing T338C are expressed in Xenopus oocytes the chemical state of the engineered cysteine can vary. Login to comment
239 The dramatic difference in reactivity toward MTS reagents may begin to explain the disparate results obtained by different laboratories with T338C CFTR (compare Refs. Login to comment
244 Altered States of T338C CFTR Are Defined by Reactivity-T338C CFTR channels in which the cysteine is in the simple thiol (or thiolate) form can be recognized by their reactivity toward mixed disulfides like MTSETϩ and diamide-GSH as well as alkylation by IAM. Login to comment
246 T338C CFTR channels in the gSX1 state were recognized initially by their reactivity toward 2-ME or DTT, a result incompatible with the simple thiol (or thiolate) form of the cysteine. Login to comment
253 It seems appropriate to view the action of 2-ME and DTT on T338C CFTRasareflectionoftheirabilitytoligandmetalsratherthantheiractivity as "reducing agents." Login to comment
266 We reported previously that single T338C CFTR channels exhibited variability in conductance consistent with different chemical states of the cysteine thiol (25). Login to comment
270 It is also common to find copper in tap water, however, so we considered the possibility that the copper that binds to T338C CFTR could originate in one or more of the solutions that bath the oocytes after they are removed from the frog. Login to comment

[hide] A possible role for intracellular GSH in spontaneo... Biometals. 2008 Jun;21(3):277-87. Epub 2007 Sep 12. Liu X
A possible role for intracellular GSH in spontaneous reaction of a cysteine (T338C) engineered into the Cystic Fibrosis Transmembrane Conductance Regulator.
Biometals. 2008 Jun;21(3):277-87. Epub 2007 Sep 12., [PMID:17849169]

Abstract [show]
The conductance of oocytes expressing T338C CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) exhibits variable responses to dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) that we proposed might be due to the extraction of copper from an adventitious binding site (Liu et al. J Biol Chem 281(12):8275-8285, 2006). In order to study the origins of variability in chemical reactivity of T338C CFTR channels, oocytes expressing T338C CFTR were exposed to BCNU (bischloroethylnitrosourea), an inhibitor of glutathione reductase. BCNU treatment caused a significant reduction of initial conductance and an increase in the response to 2-ME or DTT, suggesting a direct or indirect influence of intracellular glutathione (GSH), a major determinant of the disposition of intracellular copper. Single-channel recordings indicated that T338C CFTR channels not exposed to 2-ME or DTT exhibited multiple conductance levels not seen in T338A CFTR channels. Exposure to BCNU shifted the distribution of single-channel current amplitudes towards lower values, whereas exposure to DTT favored higher amplitudes. These results suggest that the altered chemical state of T338C channels is associated with a decreased single-channel conductance and that intracellular factors (most likely GSH) may modulate the propensity of the channel to form these altered states.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
0 A possible role for intracellular GSH in spontaneous reaction of a cysteine (T338C) engineered into the Cystic Fibrosis Transmembrane Conductance Regulator Xuehong Liu Received: 26 December 2006 / Accepted: 27 August 2007 / Published online: 12 September 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The conductance of oocytes expressing T338C CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) exhibits variable responses to dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) that we proposed might be due to the extraction of copper from an adventitious binding site (Liu et al. Login to comment
2 In order to study the origins of variability in chemical reactivity of T338C CFTR channels, oocytes expressing T338C CFTR were exposed to BCNU (bischloroethylnitrosourea), an inhibitor of glutathione reductase. Login to comment
4 Single-channel recordings indicated that T338C CFTR channels not exposed to 2-ME or DTT exhibited multiple conductance levels not seen in T338A CFTR channels. Login to comment
6 These results suggest that the altered chemical state of T338C channels is associated with a decreased single-channel conductance and that intracellular factors (most likely GSH) may modulate the propensity of the channel to form these altered states. Login to comment
9 However, the origin of the variability in the chemical reactivity among oocytes expressing T338C CFTR was not fully understood, nor was the basis for the changes in macroscopic conductance discerned, i.e., change in single-channel conductance or gating (open probability). Login to comment
10 Because intracellular GSH has been demonstrated to be an important determinant of the disposition of intracellular copper (Freedman et al. 1989; Ciriolo et al. 1990; Ascone et al. 1993; Ferreira et al. 1993), I used two-electrode-voltage-clamp (TEVC) and single-channel recording to examine the effects of changes in intracellular GSH on the spontaneous reactions of T338C CFTR. Login to comment
12 Exposure to BCNU decreased the initial conductance of oocytes expressing T338C CFTR and increased the magnitude of the response to 2-ME or DTT, as if a lower level of cellular GSH promoted the modified state of the cysteine. Login to comment
13 These results are consistent with the idea that intracellular GSH might be responsible, at least in part, for the variability in the chemical state of T338C CFTR. Login to comment
48 Results The conductance of oocytes expressing T338C CFTR and the response to 2-ME or DTT were altered by BCNU, an inhibitor of glutathione reductase GSH is the most abundant free thiol in cells and it has high affinity for metals (Rabenstein 1989). Login to comment
49 I thus considered the possibility that variable intracellular GSH concentrations might contribute to the variability in initial conductance and responses to 2-ME or DTT seen in oocytes expressing T338C CFTR (Liu et al. 2006) by altering the fractional distribution of channels containing copper. Login to comment
51 Summarized in Fig. 1 are results obtained from oocytes expressing T338C or T338A CFTRs that were either untreated, or exposed to 100 lM BCNU for 72 h prior to electrophysiological recording. Login to comment
52 Oocytes expressing T338C CFTR and exposed to 100 lM BCNU exhibited a significantly lower initial steady state conductance (22 ± 4 lS) than untreated oocytes (92 ± 14, lS, P-value \ 0.05). Login to comment
54 The conductances after exposure to 2-ME were not significantly different between treated (119 ± (( lS) and untreated oocytes expressing T338C CFTR (98 ± 13 lS). Login to comment
56 In this population, oocyte pre-exposed to BCNU did not differ Fig. 1 BCNU altered T338C CFTR conductance and its response to 2-ME. Login to comment
57 (A) The initial steady state conductance of oocytes expressing T338C CFTR (black bars) and the conductance after exposure to 1 mM 2-ME (white bars) were summarized for the control oocytes and oocytes maintained in the incubation solution (MBSH) containing 100 lM BCNU since injection of cRNA. Login to comment
62 These results are consistent with the hypothesis that BCNU- induced responses in T338C CFTR are specific to the cysteine at position 338. Login to comment
64 BCNU altered the fractional distribution of single-channel current amplitudes in oocytes expressing T338C CFTR To determine if BCNU treatment altered open probability or single-channel conductance, I recorded single-channel currents from inside-out patches detached from oocytes expressing T338C CFTR that were either untreated or exposed to BCNU. Login to comment
68 To mitigate this potential contamination by non-CFTR channels, a patch was operationally defined as containing T338C CFTR channels if the events were activated by PKA and ATP. Login to comment
70 Summarized in Fig. 2 are fractional distributions of current amplitudes extracted from patches obtained from oocytes expressing T338C CFTR at pH 7.4 (extracellular, Vm = -100 mV). Login to comment
72 We have shown previously that events with 0.6 pA amplitude represent the full conductance of T338C channels at pH 7.4 in the presence of 2-ME or DTT (Liu et al. 2004). Login to comment
79 If events with different current amplitudes represent T338C CFTR channels in different chemical states, be it oxidation or metal complexes, some of these channels might be sensitive to DTT, a strong reducing agent and a potent metal ligand (Krezel et al. 2001). Login to comment
86 We reported previously that the single-channel conductance of T338C CFTR is larger at pH 6.0 (*9 pS) than at pH 7.4 (Liu et al. 2004). Login to comment
90 To verify that a cysteine was required for the multiple current amplitudes observed in T338C CFTR, I recorded single-channel currents of T338A CFTR. Login to comment
91 The single-channel conductance of this construct is greater than that of T338C CFTR (Linsdell et al. 1998; Liu et al. 2004). Login to comment
98 Low concentration of GSH reverses sponstanous and copper-modified states at T338C locus The impact of BCNU on the chemical state of a cysteine at 338 suggests that in the event of reduction Fig. 2 BCNU altered the fractional distribution of current amplitudes of single T338C CFTR channels at pH 7.4. Login to comment
99 Fractional distribution of single-channel current amplitudes at pHextra = 7.4 from patches obtained from T338C CFTR expressing oocytes that were: (A) incubated in MBSH, (B) incubated in MBSH containing 100 lM BCNU since injection of cRNA, (C) incubated in MBSH and exposed to 10 mM DTT for about 1 to 24 hours before patching or MBSH, (D) incubated in MBSH containing 100 lM BCNU since injection of cRNA and exposed to 10 mM DTT for about 1 to 24 hours before patching. The sample current traces obtained at Vm = -100 mV for each group are shown above the bars of cytoplasmic GSH the cysteine at 338 is more likely to be chemically altered, perhaps by coordinating copper. Login to comment
102 Alternatively, efflux of GSH and GSH-conjugates via an endogenous pathway in Xenopus oocytes (Ballatori et al. 1996) may lead to a higher local concentration of GSH in the extracellular space between the plasma membrane and the follicular membrane bringing GSH into proximity with T338C. Login to comment
103 Because it is impossible at present to assay the intracellular concentration of GSH in intact cells in real time, I chose to use a functional assay to characterize the impact of externally applied GSH on the spontaneously-altered state and copper-modified state of T338C CFTR. Login to comment
104 A naive oocyte expressing T338C CFTR (Fig. 5A), was first exposed to 1 lM and then to 1 mM GSH, resulting in rapid, dose-dependent increases in conductance, similar to those seen after treatment with DTT or 2-ME, indicating a reversal of the modified state of this engineered cysteine. Login to comment
108 Under copper modified condition, exposure to 1 lM GSH and Fig. 3 BCNU altered the fractional distribution of current amplitudes of single T338C CFTR channels at pH 6.0. Login to comment
109 Fractional distribution of single-channel current amplitudes at pHextra = 6.0 from patches obtained from T338C CFTR expressing oocytes that were: (A) incubated in MBSH, (B) incubated in MBSH containing 100 lM BCNU since injection of cRNA, (C) incubated in MBSH and exposed to 10 mM DTT for about 1 to 24 hours before patching, (D) incubated in MBSH containing 100 lM BCNU since injection of cRNA and exposed to 10 mM DTT for about 1 to 24 hours before patching. The sample current traces obtained at Vm = -100 mV for each group are shown above the bars 1 mM GSH also resulted in dose-dependent increases in conductance. Login to comment
111 The similar efficacies of GSH on T338C CFTR conductance under naive and external copper-bound state strongly suggest a similar, if not identical chemical modification of T338C under the two conditions. Login to comment
112 The above result suggests that extracellular GSH can perturb copper binding at T338C locus with an affinity in the micromolar range. Login to comment
118 Results shown in Fig. 6A indicated that GSH could only partially reverse the mixed disulfide bond between T338C and MTSET+ and could do so only at a concentration nearly 1,000 fold higher than that needed to perturb the copper binding site. Login to comment
124 These experiments support the notion that GSH is an importamt determinate of chemical reactivity of T338C in naı¨ve oocytes. Login to comment
133 In addition, GSH is known to coordinate copper (Rabenstein 1989) and to be an important determinant of the disposition of intracellular copper (Freedman et al. 1989; Ciriolo et al. 1990; Ascone Fig. 5 Extracellular GSH could remove copper from T338C locus. Login to comment
134 (A) Following activation by stimulatory cocktail (10 lM Isop and 1 mM IBMX, hatched bar and crosshair), a naive oocyte expressing T338C CFTR was exposed to: 1 lM and then 1 mM GSH (open triangles), 1 mM DTT (open circles), 1 mM CuCl2 (grey circles), 1 lM GSH and 1 mM GSH, 1 mM 2-ME (open circles), (n = 4). Login to comment
137 The influence of BCNU on the 2-ME/DTT-sensitive conductance of oocyte expressing T338C CFTR suggests that decreasing cytosolic GSH increases the likelihood that copper will be bound by the adventitious metal center that is inadvertently created in the cysteine-substituted channel. Login to comment
151 (A) Following activation by stimulatory cocktail (10 lM Isop and 1 mM IBMX, hatched bar and crosshair), a naive oocyte expressing T338C CFTR was exposed to: 1 mM DTT (open circles), 1 mM MTSET+ (black circles), 100 lM GSH and 1 mM GSH (open triagles), 1 mM 2-ME (open squares). Login to comment
157 The dose-dependent response to extracellular GSH is consistent with an equilibrium mechanism in which increasing GSH shifted the equilibrium towards a state where the copper at the T338C locus was either freed or the coordination geometry was perturbed. Login to comment

[hide] Conformational changes in a pore-lining helix coup... J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3. Beck EJ, Yang Y, Yaemsiri S, Raghuram V
Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3., 2008-02-22 [PMID:18056267]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
93 Both Cd2ϩ and MTSEA had significant effects on the conductances of only five (I331C, L333C, R334C, K335C, and T338C) of the 26 Cys-substituted channels examined. Login to comment
100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to comment
127 For example, whereas L333C in the Glu1371 (WT) channel was inhibited by either Cd2ϩ or MTSEA, neither reagent was particularly effective when this mutation was present in the Gln1371 background 200 150 100 50 0 µS 15001000500 s IBMX Cd 2+ MTSEA DTT -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C Cd 2+ aM Cd 2+ bM Cd 2+ uM A B FIGURE 2. Login to comment
131 B, summary of effects of Cd2ϩ on MTSEA-modified I331C, L333C, R334C, K335C, and T338C channels. Login to comment
135 MTSEA 1371Q 600 400 200 µS 200150100500 s Cd 2+ 1371E -40 0 40 % Change in conductance I331C L333C R334C K335C T338C 1371E 1371Q * * * -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C * * * 1371Q 800 600 400 µS 2001000 s MTSEA 1371E B D E 1 2 30 s1 pAWT; Po=0.18 A 3 1 2 100 s1 pAE1371Q; Po=0.94 C FIGURE 3. Login to comment
152 In contrast, the pore-lining residues R334C and T338C exhibited very small or no differences in their functional effects in the Glu1371 and Gln1371 channels, respectively. Login to comment
153 The differences between Glu1371 and Gln1371 backgrounds in the effects of Cd2ϩ and MTSEA on I331C, L333C, R334C, K335C, and T338C channels are summarized in Fig. 3 (C and E), respectively. Login to comment
158 The cysteine residues R334C and T338C, postulated to be pore-lining residues, showed no changes in their rates of modification by either MTSEA or MTSES. Login to comment
184 Under minimal activation conditions (0.02 mM IBMX), the cysteine residues R334C, K335C, and T338C showed no significant differences in their modification rates by either MTSEA or MTSES (Fig. 6). Login to comment
197 Kinetic analyses of channel gating revealed that the decrease in open probability of MTSET-modified I331C and L333C channels was primarily because of an increase in the mean interburst duration of the A B 1.00.50.0 G0.02/ G1 I331C L333C R334C K335C T338C 200 100 0 µS 8006004002000 s 0.02 1 IBMX (mM) C -100 100 % Change in conductance I331C L333C R334C K335C T338C 0.02 mM IBMX 1 mM IBMX * * * * -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C * * * MTSEA MTSES FIGURE5.EffectsofMTSEA,andMTSESdependonCFTRactivationlevels. Login to comment
217 That study did not observe any effects of MTSEA on T338C channels, whereas we did here. Login to comment
220 However, our observations on the accessibility of R334C, K335C, and T338C and the inaccessibility of R347C are consistent with other studies (10, 11). Login to comment
223 It is possible that this mutation rather than the open 150 125 100 %G/Gi 600 s K335C I-1.0; 10 µM I-0.02; 10 µM 10 1 10 2 10 3 10 4 Modification rate (M -1 s -1 ) I331C L333C R334C K335C T338C 100 50 %G/Gi 3002001000 s I-1.0; 100 µM I-0.02;10 µM MTSEA I331CL333CR334CK335CT338C 100 75 50 25 0 %G/Gi 180120600 s I-0.02; 10 µM I-1.0; 10 µM 200 150 100 %G/Gi 120600 s I-0.02; 10 µM I-1.0; 10 µM 100 75 50 %G/Gi 3602401200 s I-1.0; 100 µM I-0.02; 10 µM 100 80 60 %G/Gi 9060300 s K335C I-1.0; 10 µM I-0.02; 10 µM 100 50 %G/Gi 180120600 s T338C I-1.0; 10 µM I-0.02; 10 µM 10 1 10 2 10 3 10 4 Modification rate (M -1 s -1 ) I331C L333C R334C K335C T338C MTSES 100 75 50 25 %G/Gi 120600 s I-1.0; 10 µM I-0.02; 10 µM 100 75 50 %G/Gi 3602401200 s I-1.0; 100 µM I-0.02; 10 µM 100 75 %G/Gi 180120600 s I-0.02; 100 µM I-1.0; 1 mM A B FIGURE 6. Login to comment
239 Furthermore, the pore-lining residues R334C and T338C showed no state-dependent changes in reactivity, which also suggests that there are no significant changes in the local electrostatic potential during channel gating. Login to comment
242 Hence, a small fraction of the increased reactivity of I331C, and L333C at low IBMX concentrations could be due to a relief from this block, although such an increase in reactivity is not observed for R334C and T338C. Login to comment

[hide] State-dependent access of anions to the cystic fib... J Biol Chem. 2008 Mar 7;283(10):6102-9. Epub 2007 Dec 31. Fatehi M, Linsdell P
State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2008 Mar 7;283(10):6102-9. Epub 2007 Dec 31., 2008-03-07 [PMID:18167343]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN)(2)(-), to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
74 In fact, similar charge-dependent effects were observed in R334C, K335C, T338C, and S341C (Fig. 3). Login to comment
114 F, wild type (both panels); E, R334C (left); Ⅺ, K335C (left); ‚, F337C (right); ƒ, T338C (right); छ, S341C (right) (mean of data from 3-9 patches). Login to comment
117 We therefore used Au(CN)2 - , a highly permeant anion that has been shown to covalently modify the introduced cysteine in T338C-CFTR to block the permeation pathway (13). Login to comment
119 We therefore reasoned that if Au(CN)2 - entered the pore to modify the introduced cysteine of T338C covalently, only very small currents could be activated by PKA, ATP, and PPi but that currents would subsequently be activated on exposure to CN- . Login to comment
123 In T338C, however, pretreatment with the same concentration of Au(CN)2 - for only 1 min led to the appearance of a KCN-sensitive component of current (Fig. 7A). Login to comment
128 Thus, although it appears that Au(CN)2 - can modify T338C-CFTR with or without cAMP stimulation, the dramatic increase in the proportion of channels apparently modified by Au(CN)2 - when cAMP stimulation is applied concurrently with Au(CN)2 - exposure suggests that the rate of modification is far greater in activated channels than in nonactivated channels. Login to comment
140 Conformational Change in the Pore on Activation of CFTR 6106 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 10•MARCH 7, each of R334C, K335C, and S341C, like T338C, the apparent degree of Au(CN)2 - modification as determined by the KCN- sensitive component of the current was significantly enhanced by cAMP stimulation (Fig. 7E). Login to comment
179 Example I-V relationships recorded following pretreatment with 10 ␮M KAu(CN)2 for 5 min (wild type) or 1 min (T338C) is shown. Login to comment
183 The asterisk indicates a significant differencefromcontrolconditionsforthesamechannelvariant(pϽ0.00001).C,KCN-inducedchangesinCFTR macroscopic conductance for individual patches expressing T338C-CFTR, following pretreatment with KAu(CN)2 alone (E) or KAu(CN)2 plus forskolin and IBMX (F) are shown. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2009 Oct 27;48(42):10078-88. Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC
Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore.
Biochemistry. 2009 Oct 27;48(42):10078-88., 2009-10-27 [PMID:19754156]

Abstract [show]
The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
52 We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment
153 The reactivity of F337C/wt CFTR toward the channel-permeant probes, although similar to that seen previously with T338C/wt CFTR (12), differed significantly in detail. Login to comment
155 After inhibition of F337C conductance by [Au- (CN)2]- , exposure of oocytes to a competing thiol, 2-ME, did not reverse the inhibition of conductance as previously seen with T338C/wtCFTR(12), butthe inhibition wasrelievedbyexposing the oocyte to a solution containing 1 mM KCN as expected from the high-affinity liganding of Au(1) by the cyanide anion (12). Login to comment

[hide] Dual roles of the sixth transmembrane segment of t... J Gen Physiol. 2010 Sep;136(3):293-309. Bai Y, Li M, Hwang TC
Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.
J Gen Physiol. 2010 Sep;136(3):293-309., [PMID:20805575]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
82 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment
107 Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT). Login to comment

[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9]. Login to comment

[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]

Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
248 However, because the mutations T338C and T339C did not react with MTS reagents, the side PKA phosphorylation but did not substitute for ATP in opening phosphorylated CFTR Cl0 channels.chains of these residues do not interact with permeating ions (31, 77). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7. Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O'Donnell N, Dawson DC, Sansom MS
Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore.
Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7., [PMID:22352759]

Abstract [show]
We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
367 Similarly, while both extracellular MTSET+ and MTSES- react with a cysteine at position 338, the ratio of the rates of reaction (kMTSET +/kMTSES -) of these oppositely charged reagents was <1.0 for T338C/wt CFTR and >1.0 for T338C/ R334D CFTR as expected if the charge at this position makes a major contribution to the electrostatic potential at the outer rim of the bottleneck.40 Studies of the impact of covalent and noncovalent modifications at position 338 also argue that the electrostatic potential at this site, just on the outward-facing lip of the bottleneck, is critical for anion conduction. Login to comment

[hide] Locating a Plausible Binding Site for an Open Chan... Mol Pharmacol. 2012 Aug 24. Norimatsu Y, Ivetac A, Alexander C, O'Donnell N, Frye L, Sansom MS, Dawson DC
Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator.
Mol Pharmacol. 2012 Aug 24., [PMID:22923500]

Abstract [show]
High-throughput screening has led to the identification of small-molecule blockers of the CFTR chloride channel, but the structural basis of blocker binding remains to be defined. We recently developed molecular models of the CFTR channel based on homology to the bacterial transporter, Sav1866, that could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker like GlyH-101, thought to act by entering the channel from the extracellular side. As a more stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual ligand docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest scoring, docked position was near two pore-lining residues, F337 and T338, and the rate of reaction of anionic thiol-directed reagents with cysteines substituted at either of these positions was slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (F342) was replaced with alanine, the apparent affinity of the blocker increased by approximately 200 fold. A Molecular Mechanics-Generalized Born/Surface Area (MM-GB/SA) analysis of GlyH-101 binding predicted that substitution of F342 with alanine would substantially increase blocker affinity, primarily due to decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
115 Figure 3C and 3D contain the time courses for the reactions of [Au(CN)2]- with F337C and T338C CFTR pre-and post-activation. Login to comment
116 In the case of T338C CFTR, different concentrations of [Au(CN)2]- were used to construct the time course (50 µM for the pre-activation rate; 5 µM for the post-activation rate), in order to compensate for the slower reaction rate in the pre-activation condition. Login to comment
138 Figure 5A contains a representative time course showing the cumulative effect of 30 s and 1 min applications of 5 µM [Au(CN)2]- to an oocyte expressing T338C CFTR. Login to comment
140 Inspection of the response to the first, 30s exposure reveals that the extent of modification of T338C conductance by [Au(CN)2]- was markedly reduced when the reagent was applied in the presence of the blocker. Login to comment
141 Figure 5C and 5D summarize the inhibition of F337C CFTR and T338C CFTR by [Au(CN)2]- in the presence and in the absence of GlyH-101. Login to comment
144 In Figure 5E and 5F the measured second order rate constants for covalent modification of F337C and T338C CFTR are plotted versus GlyH-101 concentration. Login to comment
148 As a test for the role of charge-charge interactions we compared the protection of T338C CFTR against reaction with MTSET+ and MTSES- . Login to comment
149 The results summarized in Figure 6 show that T338C is protected by GlyH-101 from negatively-charged MTSES- but not from positively-charged MTSET+ , indicating that the charge on the blocker is a major determinant of the protection effect at this position. Login to comment
174 We analyzed GlyH-101 binding to wt and F342A CFTR channels using the MM-GB/SA method as described by Guimaraes and Cardozo, (2008). Login to comment
209 Second, alkylation of T338C CFTR with IAM, which results in covalent addition of an acetamide moiety predicted by the model to create a steric clash with GlyH-101, significantly reduced the apparent binding affinity of GlyH-101. Login to comment
210 In contrast, alkylation of F337C CFTR with IAM is not predicted by the molecular model to cause a steric clash and does not markedly alter GlyH-101 block. Login to comment
211 Finally, when a predicted steric clash between the side chain of F342 and the naphthalene tail of the bound blocker was removed by mutating the residue to an alanine (F342A), the apparent blocker affinity was increased by more than 200-fold, a change that was consistent with free energies of binding estimated using a MM-GB/SA approach. Login to comment
220 The state-dependent reactivity of T338C CFTR observed in the current study is consistent with the finding of Beck et al., (2008) that MTSES- reacts slightly faster with a high open probability mutant T338C/E1371Q CFTR than with T338C/wt CFTR.2 Mornon et al., (2009) created a homology model of CFTR based on the inward-facing conformation of a prokaryotic transporter, MsbA (Ward et al., 2007) (PDB code: 3B5X). Login to comment
224 A conformational change of this sort would be consistent with the state-dependent reactivity of F337C and T338C CFTR observed in the current study. Login to comment
225 The MsbA-based model of Mornon et al., (2009) also predicts that the side chain of R334 protrudes into the external aqueous environment, and when R334 is mutated to a cysteine in the MsbA-based model of Mornon et al., (2009) using Maestro (version 9.1, Schrödinger LLC), the reactive thiolate is clearly accessible from the extracellular solution (Figure 9C), consistent with the closed state reactivity of R334C observed in the current study and by Zhang et al., (2005). Login to comment
226 On the other hand, the mechanism that renders R334C CFTR unreactive in the conducting state of CFTR is not clear. Login to comment
127 When either Phe337 or Thr338 was replaced with cysteine, the EC50 for GlyH-101 blockade at 0 mV was only modestly increased (Table 1).1 Increasing the bulk of either cysteine side chain through alkylation with iodoacetamide further increased the EC50(0) b03;6-fold for position 338 but only 1.3-fold for position 337. Login to comment
158 Figure 3, C and D, contains the time courses for the reactions of [Au(CN)2]afa; with the F337C and T338C CFTRs before TABLE 1 EC50 at 0 mV (mean afe; S.E.M.) for GlyH-101 for wt and mutant CFTRs, with and without modification with iodoacetamide CFTR EC50 at 0 mV òe;M wt 1.1 afe; 0.11 (n afd; 4) K95C 1.4 afe; 0.35 (n afd; 4) F337C 1.8 afe; 0.06 (n afd; 3) F337C af9; iodoacetamide 2.4 afe; 0.29 (n afd; 3) T338C 3.7 afe; 0.27 (n afd; 3) T338C af9; iodoacetamide 24 afe; 2.6 (n afd; 3) Fig. 3. Login to comment
163 C and D, time courses of the decreases in normalized conductance as a result of F337C (C) and T338C (D) modifications with [Au(CN)2]afa; . Login to comment
166 For the T338C CFTR, the abscissa represents cumulative [Au(CN)2]afa; exposure (exposure time afb; [Au(CN)2]afa; concentration used). Login to comment
168 The rate for the T338C CFTR was almost 30 times slower before activation. Login to comment
172 The second-order reaction rate constants for the T338C CFTR before and after activation were 1.2 afb; 102 and 3.0 afb; 103 Mafa;1 safa;1 , respectively. Login to comment
204 and 1-min applications of 5 òe;M [Au(CN)2]afa; to an oocyte expressing T338C CFTR. Login to comment
205 A similar procedure, in which [Au(CN)2]afa; was applied during brief exposures of the oocyte to GlyH-101 and the extent of the reaction was estimated from the conductance recovered after washout of the blocker, is illustrated in Fig. 5B. Inspection of the response to the first, 30-s exposure revealed that the extent of modification of T338C CFTR conductance by [Au(CN)2]afa; was markedly reduced when the reagent was applied in the presence of the blocker. Login to comment
206 Figure 5, C and D, summarizes the inhibition of the F337C and T338C CFTRs by [Au(CN)2]afa; in the presence and absence of GlyH-101. Login to comment
215 C and D, F337C (C) and T338C (D) CFTR channels were protected by 10 òe;M GlyH-101 from reactions with [Au(CN)2]afa; . Login to comment
217 The F337C CFTR was reacted with 600 òe;M [Au(CN)2]afa; and the T338C CFTR was reacted with 5 òe;M [Au(CN)2]afa; in the presence and absence of 10 òe;M GlyH-101. Login to comment
231 As a test for the role of charge-charge interactions, we compared the protection of the T338C CFTR against reactions with MTSETaf9; and MTSESafa; . Login to comment
232 The results summarized in Fig. 6 showed that the T338C CFTR was protected by GlyH-101 from negatively charged MTSESafa; but not from positively charged MTSETaf9; , indicating that the charge on the blocker is a major determinant of the protective effect at this position. Login to comment
250 GlyH-101 protection of T338C (an engineered cysteine at position 338) from MTSESafa; but not from MTSETaf9; . Login to comment
251 A, the T338C CFTR was reacted with 5 òe;M MTSESafa; in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment
255 The second-order reaction rate constants for MTSESafa; in the presence and absence of 10 òe;M GlyH-101 were 1.2 afb; 103 and 3.3 afb; 103 Mafa;1 safa;1 , respectively. B, the T338C CFTR was reacted with 50 òe;M MTSETaf9; in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment
317 Second, alkylation of the T338C CFTR with iodoacetamide, which results in covalent addition of an acetamide moiety that is predicted by the model to create a steric clash with GlyH-101, significantly reduced the apparent binding affinity of GlyH-101. Login to comment
346 The state-dependent reactivity of the T338C CFTR that was observed in the current study is consistent with the finding by Beck et al. (2008) that MTSESafa; reacted slightly faster with a mutant with high open probability (T338C/ E1371Q CFTR) than with the T338C/wt CFTR.2 Mornon et al. (2009) created a homology model of the CFTR that was based on the inward-facing conformation of the prokaryotic transporter MsbA (PDB code 3B5X) (Ward et al., 2007). Login to comment
350 A conformational change of this sort would be consistent with the state-dependent reactivity of the F337C and T338C CFTRs observed in the current study. Login to comment
352 Wang and Linsdell (2012) studied reactions of the T338C/E1371Q CFTR with MTSESafa; and [Au(CN)2]afa; and suggested that the reaction of an engineered cysteine at position 338 with externally applied reagents was favored in the closed state. Login to comment
353 However, this observation conflicts with that by Beck et al. (2008), who reported that MTSESafa; reacted slightly faster with a double-mutant with high open probability (T338C/E1371Q CFTR) than with the T338C/wt CFTR. Login to comment
354 We observed reaction rates for MTSESafa; with T338C/wt and T338C/E1371Q CFTRs that were similar to those observed by Beck et al. (2008) (data not shown), which supports the idea that the T338C CFTR reacts in the open state. Login to comment

[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
43 In the present work, we have compared changes in the accessibility of T338C in TM6 with L102C in TM1 to both intracellular and extracellular cysteine-reactive reagents, both large, impermeant [2-sul- fonatoethyl] MTS (MTSES) and smaller, permeant Au(CN)2 - ions, under conditions in which ATP-dependent channel gating is altered. Login to comment
50 Two reporter cysteines in the pore were studied: T338C in TM6, which is modified by both intracellular and extracellular MTS reagents (17), and L102C in TM1, which is modified by intracellular, but not extracellular MTS reagents (18). Login to comment
52 These two reporter cysteine substitutions were combined with mutations in the NBDs that affect ATP-dependent channel gating: K464A (NBD1) and E1371Q (NBD2). Login to comment
79 Fig. 1 shows the influence of these NBD mutations on the rate of modification of two cysteines introduced deep into the channel pore from the inside, T338C in TM6 and L102C in TM1. Login to comment
82 Rate of modification of T338C and L102C by internal MTSES. Login to comment
86 The decline in current amplitude following MTSES application has been fitted by a single exponential function. C, average modification rate constants (k) for MTSES, calculated from fits to data such as those shown in A and B. Asterisks indicate a significant difference from the cysteine mutants T338C and L102C (black bars) (p Ͻ 0.05). Login to comment
89 For modification of T338C, the mean modification rate constant was decreased ϳ2.4-fold in a K464A background and increased ϳ3.9-fold in E1371Q, whereas the modification rate constant for L102C was decreased by ϳ26% in K464A and increased ϳ2.0-fold in E1371Q. Login to comment
90 As in our previous work (22), the effects of the E1371Q mutation were mimicked by locking channels open by treatment with 2 mM pyrophosphate (data not shown; ϳ3.9-fold increase for T338C and ϳ1.6-fold increase for L102C). Login to comment
92 To investigate whether permeant anions show the same regulated access from the cytoplasm to T338C and L102C, we investigated channel modification by Au(CN)2 - , a highly permeant anion that has been used previously to modify cysteine side chains in the CFTR pore (14, 22). Login to comment
93 As shown in Fig. 2, application of a low concentration of Au(CN)2 - (2 ␮M) caused a rapid inhibition of current carried by both T338C and L102C. Login to comment
94 As with MTSES, the rate of modification by Au(CN)2 - was significantly decreased by the K464A mutation (by ϳ1.8-fold for T338C and ϳ3.4-fold for L102C) and significantly increased by the E1371Q mutation (by ϳ5.6-fold for T338C and ϳ1.8-fold for L102C), as well as by pyrophosphate treatment (by ϳ6.0-fold for T338C and ϳ2.0-fold for L102C; data not shown). Login to comment
96 Regulated Access from Extracellular Solution to Pore- T338C is modified not only by intracellular, but also by extracellular MTS reagents (14, 17, 26), whereas L102C was reported to be insensitive to extracellular MTS reagents (18). Login to comment
98 Expression of all CFTR constructs (except those containing the E1371Q mutation, see below) in baby hamster kidney cells led to the appearance of cAMP-activated whole cell currents that were inhibited by the specific CFTR inhibitor GlyH-101 (Fig. 3 and supplemental Fig. S2) and which were not observed in cells transfected with vector alone (supplemental Fig. S2). Login to comment
99 Expression of all E1371Q-CFTR constructs led to the appearance of constitutive, cAMP-insensitive but GlyH-101-inhibited whole cell currents (supplemental Fig. S2). Login to comment
101 In contrast, T338C was strongly inhibited by very much lower concentrations of MTSES (1 ␮M) and Au(CN)2 - (200 nM) (Fig. 3B). Login to comment
104 Fig. 4A shows examples of the rate of current inhibition in response to application of a common concentration of MTSES (1 ␮M) in T338C, T338C/K464A, and T338C/E1371Q. Login to comment
109 The decline in current amplitude following Au(CN)2 - application has been fitted by a single exponential function. C, average modification rate constants(k)forAu(CN)2 - ,calculatedfromfitstodatasuchasthoseshowninAandB.AsterisksindicateasignificantdifferencefromthecysteinemutantsT338C and L102C (black bars) (p Ͻ 0.02). Login to comment
110 Data are mean from three or four patches. Alternating Access Model of CFTR MARCH 23, 2012ȂVOLUME 287•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 10159 fication (Fig. 4B) suggests an increase of ó3.7-fold in T338C/ K464A and a dramatic decrease of ϳ35-fold in T338C/E1371Q compared with T338C alone. Login to comment
111 Note that, because MTSES modification was so slow in T338C/E1371Q, the rate constant for modification of this construct was calculated from experiments using a higher concentration of MTSES (200 ␮M). Login to comment
113 Fig. 5 shows a similar analysis of the rate of modification by extracellular Au(CN)2 - , both for T338C (Fig. 5A; 200 nM Au(CN)2 - ) and for L102C (Fig. 5B; 10 ␮M Au(CN)2 - ). Login to comment
114 Quantification of the rate constant for modification (Fig. 5C) suggests, for modification of T338C, an increase of ϳ5.7-fold in K464A and a decrease of ϳ150-fold in E1371Q, and for modification of L102C, an increase of ϳ2.3-fold in K464A and a decrease of ϳ2.7-fold in E1371Q. Login to comment
115 As with extracellular MTSES modification of T338C/E1371Q (Fig. 4), the rate constant for Au(CN)2 - modification of E1371Q channels was calculated from experiments using higher concentrations of Au(CN)2 - (100 ␮M). Login to comment
116 Changing Patterns of Accessibility Suggest Marker Cysteine Residues "Switch Sides" of Membrane during Gating-The effects of NBD mutations on the rate of modification of T338C and L102C by internal cysteine-reactive reagents (estimated from experiments on inside-out membrane patches) and by external cysteine-reactive reagents (estimated from whole cell current recording experiments) are compared in Fig. 6. Login to comment
120 In each panel, it can be seen that the rate of modification by internal MTSES and Au(CN)2 - increases in the order K464A Ͻ Cys-less Ͻ E1371Q, whereas modification by extracellular MTSES (in T338C) and Au(CN)2 - shows the opposite pattern, K464A Ͼ Cys-less Ͼ E1371Q. Login to comment
122 This suggests that the reporter cysteines in the pore that we have used, T338C and L102C, are capable of "moving" from a relatively internally accessible position to a relatively externally accessible position. Login to comment
127 Sample whole cell currents were recorded at ϩ30 mV for Cys-less (A), T338C (B), and L102C (C). Login to comment
130 B, T338C currents were inhibited by low concentrations of MTSES (1 ␮M) or Au(CN)2 - (200 nM). Login to comment
141 Previously our group showed that T338C could be modified by both intracellular and extracellular MTS reagents, even though these reagents are thought to be too large to permeate through the channel pore (17). Login to comment
143 Thus, it is possible that T338C is modified by intracellular MTSES in open channels and by extracellular MTSES in closed channels; the measured apparent rate of modification would then be dependent on the intrinsic rate of modification and the proportion of time the channel spends in the open and closed state in different channel constructs. Login to comment
147 However, these cysteines in the outer pore region (L333C, R334C, and K335C) are not modified by intracellular MTS reagents under any conditions (17, 27), suggesting that unlike T338C they cannot move to a position that is accessible to large cytoplasmic substances. Login to comment
152 L102C, like T338C, becomes apparently more accessible to internal cysteine reactive reagents in open channels (Fig. 6B), but is inaccessible to extracellular MTSES (Fig. FIGURE 4. Login to comment
153 Rate of modification of T338C by external MTSES. Login to comment
157 Modification rate constant for T338C/E1371Q was quantified from experiments using a higher concentration of MTSES (200 ␮M). Login to comment
158 Asterisks indicate a significant difference from T338C alone (p Ͻ 0.0005). Login to comment
160 L102C is accessible to permeant Au(CN)2 - ions applied to either side of the membrane, as expected for a permeant probe that ought to access the entire permeation pathway, and as with T338C access from the outside decreases as access from the inside increases (Fig. 6D), again consistent with easier access from the cytoplasm in open channels and from the extracellular solution in closed channels. Login to comment
161 One possible explanation for the difference in external accessibility of L102C in TM1 and T338C in TM6 is that T338C is located in a more superficial position in the outer mouth of the pore (at least in closed channels), such that it can be accessed by large extracellular MTS reagents that cannot penetrate further into the pore from the outside to modify L102C (Fig. 7A). Login to comment
162 Consistent with this differential access from the outside, the rate of modification by extracellular Au(CN)2 - is approximately 35 times greater for T338C than for L102C in a Cys-less background (Fig. 5C). Login to comment
163 One apparent problem with this explanation is that a residue only slightly closer to the outer end of TM1 (R104C) is accessible to extracellular, but not intracellular MTS reagents (see above); the model shown in Fig. 7A should put this residue close to Thr-338. Login to comment
170 Rate of modification of T338C and L102C by external Au(CN)2 - . Login to comment
174 Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)2 - (100 ␮M). Login to comment
175 Asterisks indicate a significant difference from T338C and L102C alone as applicable (black bars) (p Ͻ 0.01).Data are mean from three or four patches. Login to comment
178 Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)2 - (C and D) applied to the intracellular (●, inside) or extracellular (E, outside) side of the membrane. Login to comment
191 In this model, reduced access from the extracellular solution in open channels is due to partial closure of a vestigial open gate, which decreases the rate of entry of extracellular MTSES and Au(CN)2 - to T338C and L102C, although not completely occluding the pore and thus allowing Cl- permeation. Login to comment
199 The dramatic decrease in modification rate for external MTSES and Au(CN)2 - seen in T338C/E1371Q (Figs. 4B, 5C, and 6) suggests that access to this narrow region from the extracellular solution is greatly decreased in open channels. Login to comment
201 FIGURE7.ModelsofCFTRporestructureduringgating.Theimplicationsof the current experimental findings, that T338C and L102C in the CFTR pore show increased access to the extracellular solution in the closed state and increased access to the intracellular solution in the open state, can be interpreted according to a number of different simple diagram models of channel function. Login to comment
45 In the present work, we have compared changes in the accessibility of T338C in TM6 with L102C in TM1 to both intracellular and extracellular cysteine-reactive reagents, both large, impermeant [2-sul- fonatoethyl] MTS (MTSES) and smaller, permeant Au(CN)2 afa; ions, under conditions in which ATP-dependent channel gating is altered. Login to comment
85 Rate of modification of T338C and L102C by internal MTSES. Login to comment
95 As in our previous work (22), the effects of the E1371Q mutation were mimicked by locking channels open by treatment with 2 mM pyrophosphate (data not shown; b03;3.9-fold increase for T338C and b03;1.6-fold increase for L102C). Login to comment
97 To investigate whether permeant anions show the same regulated access from the cytoplasm to T338C and L102C, we investigated channel modification by Au(CN)2 afa; , a highly permeant anion that has been used previously to modify cysteine side chains in the CFTR pore (14, 22). Login to comment
106 In contrast, T338C was strongly inhibited by very much lower concentrations of MTSES (1 òe;M) and Au(CN)2 afa; (200 nM) (Fig. 3B). Login to comment
117 Note that, because MTSES modification was so slow in T338C/E1371Q, the rate constant for modification of this construct was calculated from experiments using a higher concentration of MTSES (200 òe;M). Login to comment
119 Fig. 5 shows a similar analysis of the rate of modification by extracellular Au(CN)2 afa; , both for T338C (Fig. 5A; 200 nM Au(CN)2 afa; ) and for L102C (Fig. 5B; 10 òe;M Au(CN)2 afa; ). Login to comment
121 As with extracellular MTSES modification of T338C/E1371Q (Fig. 4), the rate constant for Au(CN)2 afa; modification of E1371Q channels was calculated from experiments using higher concentrations of Au(CN)2 afa; (100 òe;M). Login to comment
126 In each panel, it can be seen that the rate of modification by internal MTSES and Au(CN)2 afa; increases in the order K464A b0d; Cys-less b0d; E1371Q, whereas modification by extracellular MTSES (in T338C) and Au(CN)2 afa; shows the opposite pattern, K464A b0e; Cys-less b0e; E1371Q. Login to comment
128 This suggests that the reporter cysteines in the pore that we have used, T338C and L102C, are capable of "moving" from a relatively internally accessible position to a relatively externally accessible position. Login to comment
133 Sample whole cell currents were recorded at af9;30 mV for Cys-less (A), T338C (B), and L102C (C). Login to comment
136 B, T338C currents were inhibited by low concentrations of MTSES (1 òe;M) or Au(CN)2 afa; (200 nM). Login to comment
149 Thus, it is possible that T338C is modified by intracellular MTSES in open channels and by extracellular MTSES in closed channels; the measured apparent rate of modification would then be dependent on the intrinsic rate of modification and the proportion of time the channel spends in the open and closed state in different channel constructs. Login to comment
159 Rate of modification of T338C by external MTSES. Login to comment
164 Asterisks indicate a significant difference from T338C alone (p b0d; 0.0005). Login to comment
167 L102C is accessible to permeant Au(CN)2 afa; ions applied to either side of the membrane, as expected for a permeant probe that ought to access the entire permeation pathway, and as with T338C access from the outside decreases as access from the inside increases (Fig. 6D), again consistent with easier access from the cytoplasm in open channels and from the extracellular solution in closed channels. Login to comment
168 One possible explanation for the difference in external accessibility of L102C in TM1 and T338C in TM6 is that T338C is located in a more superficial position in the outer mouth of the pore (at least in closed channels), such that it can be accessed by large extracellular MTS reagents that cannot penetrate further into the pore from the outside to modify L102C (Fig. 7A). Login to comment
169 Consistent with this differential access from the outside, the rate of modification by extracellular Au(CN)2 afa; is approximately 35 times greater for T338C than for L102C in a Cys-less background (Fig. 5C). Login to comment
177 Rate of modification of T338C and L102C by external Au(CN)2 d1a; . Login to comment
181 Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)2 afa; (100 òe;M). Login to comment
182 Asterisks indicate a significant difference from T338C and L102C alone as applicable (black bars) (p b0d; 0.01).Data are mean from three or four patches. Login to comment
185 Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)2 afa; (C and D) applied to the intracellular (cf;, inside) or extracellular (E, outside) side of the membrane. Login to comment
198 In this model, reduced access from the extracellular solution in open channels is due to partial closure of a vestigial open gate, which decreases the rate of entry of extracellular MTSES and Au(CN)2 afa; to T338C and L102C, although not completely occluding the pore and thus allowing Clafa; permeation. Login to comment
206 The dramatic decrease in modification rate for external MTSES and Au(CN)2 afa; seen in T338C/E1371Q (Figs. 4B, 5C, and 6) suggests that access to this narrow region from the extracellular solution is greatly decreased in open channels. Login to comment
208 FIGURE7.ModelsofCFTRporestructureduringgating.Theimplicationsof the current experimental findings, that T338C and L102C in the CFTR pore show increased access to the extracellular solution in the closed state and increased access to the intracellular solution in the open state, can be interpreted according to a number of different simple diagram models of channel function. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2011 Nov 29;50(47):10311-7. Epub 2011 Nov 4. Liu X, Dawson DC
Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway.
Biochemistry. 2011 Nov 29;50(47):10311-7. Epub 2011 Nov 4., [PMID:22014307]

Abstract [show]
Cysteine scanning has been widely used to identify pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR). These studies, however, have been typically conducted at room temperature rather than human body temperature. Reports of substantial effects of temperature on gating and anion conduction in CFTR channels as well as an unexpected pattern of cysteine reactivity in the sixth transmembrane segment (TM6) prompted us to investigate the effect of temperature on the reactivity of cysteines engineered into TM6 of CFTR. We compared reaction rates at temperatures ranging from 22 to 37 degrees C for cysteines placed on either side of an apparent size-selective accessibility barrier previously defined by comparing reactivity toward channel-permeant and channel-impermeant, thiol-directed reagents. The results indicate that the reactivity of cysteines at three positions extracellular to the position of the accessibility barrier, 334, 336, and 337, is highly temperature-dependent. At 37 degrees C, cysteines at these positions were highly reactive toward MTSES(-), whereas at 22 degrees C, the reaction rates were 2-6-fold slower to undetectable. An activation energy of 157 kJ/mol for the reaction at position 337 is consistent with the hypothesis that, at physiological temperature, the extracellular portion of the CFTR pore can adopt conformations that differ significantly from those that can be accessed at room temperature. However, the position of the accessibility barrier defined empirically by applying channel-permeant and channel-impermeant reagents to the extracellular aspect of the pore is not altered. The results illuminate previous scanning results and indicate that the assay temperature is a critical variable in studies designed to use chemical modification to test structural models for the CFTR anion conduction pathway.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
85 Figures 2-4 contain the results of similar experiments conducted with oocytes expressing R334C, I336C, and T338C CFTR channels. Login to comment
88 The reaction of MTSES- with T338C CFTR was the most rapid at 22 °C and was nevertheless increased at 37 °C. Login to comment
93 As expected, the apparent activation energies varied widely, ranging from being that expected for disulfide exchange15,16 for T338C CFTR to values in the range of those generally associated with protein conformational change.17-20 Figure 6 summarizes the inhibition by MTSES- of CFTR conductance at 22 °C (27 °C for F337C CFTR) and 37 °C in oocytes expressing CFTR constructs bearing substituted cysteines at positions extracellular to (334 and 336-338) and cytoplasmic to (339-342 and 344) the apparent accessibility cutoff defined by Alexander et al.10 It is apparent that, despite the dramatic increases in the reaction rates of cysteines extracellular to the cutoff, the position of the cutoff was unchanged at 37 °C. Login to comment
97 Temperature Dependence of MTSES- Modification kMTSES (M-1 s-1 ) mutant 22 °C 30 °C 32 °C 37 °C Ea (kJ/mol) R334C 2648 ± 259 (n = 3) 9411 ± 1210 (n = 5) 18407 ± 3240 (n = 3) 98 I336C 1.2a 2.3 ± 0.1 (n = 3) 6.9 ± 0.4 (n = 4) 88 F337C 2.6 ± 0.7 (27 °C)b (n = 3) 5.1 ± 1.2 (n = 3) 19.4 ± 4.4 (n = 4) 157 T338C 4067 ± 573 (n = 5) 7192 ± 370 (n = 4) 7972 ± 1019 (n = 6) 35 a Value from ref 10. b The reaction rate was undetectable at 22 °C, so the value determined at 27 °C was used. Login to comment
100 Increased temperature increased the rate of reaction of T338C CFTR with MTSES- . Login to comment
101 An oocyte expressing T338C CFTR was activated using a stimulatory cocktail and then (A) exposed to 100 μM DDTC to reverse spurious thiol reactions (white bar and circles). Login to comment
102 Exposure to 5 μM MTSES- (dark gray bar and circles) profoundly inhibited the T338C CFTR conductance. Login to comment
106 Arrhenius plots for (A) R334C, (B) I336C, (C) F337C, and (D) T338C CFTR. Login to comment

[hide] Alignment of transmembrane regions in the cystic f... J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11. Wang W, El Hiani Y, Linsdell P
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11., [PMID:21746847]

Abstract [show]
Different transmembrane (TM) alpha helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
139 Our work concerning intracellular MTS reagent modification in TM6 also identified some cysteines that could be modified in both activated and nonactivated channels (e.g., V345C and M348C), and others that could apparently be modified only after channel activation (e.g., T338C, S341C, and I344C), suggesting a state-dependent conformational change that alters access of internally applied MTS reagents into the pore (El Hiani and Linsdell, 2010). Login to comment
227 Second, whereas cysteines substituted for TM6 residues in the putative narrow pore region-F337C, T338C, and S341C-could be modified by both intracellular and extracellular MTS reagents (El Hiani and Linsdell, 2010), no residues that could be modified from both sides of the membrane were identified in TM1. Login to comment
256 For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ). Login to comment

[hide] CFTR: a cysteine at position 338 in TM6 senses a p... Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10. Liu X, Zhang ZR, Fuller MD, Billingsley J, McCarty NA, Dawson DC
CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore.
Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10., [PMID:15361410]

Abstract [show]
We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
1 Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. Login to comment
2 The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. Login to comment
4 The relative rates of chemical modification of T338C CFTR by MTSET1 and MTSESÿ were also altered by the charge at 334. Login to comment
53 In studies of T338C CFTR, the pipette solution contained 196 mM NMDG-Cl, 2 mM MgCl2, and 5 mM HEPES HemiNa, pH 7.4; 5 mM MES was used in place of HEPES when a pH of 6 was desired for the pipette solution. Login to comment
69 Recordings of single, T338C channels in experiments involving covalent modification posed additional challenges for two reasons. Login to comment
72 Finally, preliminary experiments indicated that, in contrast to results obtained previously with R334C CFTR, reaction of T338C channels with MTSET1 actually reduced single-channel conductance, rendering the resulting single-channel currents more difficult to discriminate from the small (0.1-0.3 pA) background Clÿ channels sometimes seen in patches from oocytes. Login to comment
79 Recording conditions were designed to maximize the likelihood of recording T338C single channels in each of three states: unmodified; MTSET1 modified; and MTSET1 modified, but subsequently exposed to 2-ME to reverse the modification (see text). Login to comment
92 We interpreted the titration behavior of the conductance due to T338C CFTR as reflecting the pH-dependent change in the time-averaged, partial negative charge on any single thiolate anion. Login to comment
94 For ease of comparison, in T338C/R334X (X ¼ A or E) CFTRs and T338H/R334C CFTRs in which the cysteine was always blocked by reaction with MTS reagents or NEM, the titration curves were expressed in a normalized form. Login to comment
103 RESULTS The conductance of oocytes expressing T338C CFTR is highly sensitive to changes in bath pH Testing the hypothesis that a cysteine at position 338 resides within the conduction pathway required that we alter the charge at this locus. Login to comment
104 In previous experiments using R334C CFTR we found that charge change could be effected by means of covalent modification with thiol-directed reagents like MTSET1 or MTSESÿ , or by using changes in bath pH to alter the partial negative charge on the thiolate anion (Smith et al., 2001). Login to comment
106 Fig. 2 A contains a plot of gCl at Vm ¼ Erev versus time from a representative experiment (n ¼ 5) designed to test the pH-sensitivity of T338C CFTR. Login to comment
113 It can be seen that as the bath pH became more acidic the I-V plot changed in two ways: gCl at Vm ¼ Erev FIGURE 2 The pH-induced change in conductance of oocytes expressing T338C CFTR. Login to comment
120 (B) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338C CFTR. Login to comment
121 (C) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338A CFTR. Login to comment
124 Changing the bath pH had essentially no effect on the conductances of oocytes expressing T338A CFTR (Fig. 2 C, n ¼ 3), nor did the same maneuver alter the conductances of oocytes expressing T338S (Fig. 3) or wt CFTR (Smith et al., 2001), consistent with the idea that the pH-dependent change in conductance of T338C CFTR was due to the titration of the cysteine substituted at 338. Login to comment
125 As an additional test of the hypothesis that the pH-induced response seen in T338C CFTR was due to the titration of the engineered cysteine, we exposed oocytes expressing T338C CFTR to NEM, a reagent that forms a thioether bond with the cysteine, and thereby blocks titration of the thiol group. Login to comment
126 As summarized in Fig. 3, acidifying the bathing solution (pH 7.4-6) induced an ;68% (612) increase in conductance in oocytes expressing T338C CFTR whereas alkalinizing the bath (pH 7.4-9) decreased the conductance by ;80% (64). Login to comment
128 Pre-exposure to 1 mM MTSET1 or 1 mM MTSESÿ also prevented the response of T338C CFTR conductance to a change in bath pH (X. Liu and D. C. Dawson, unpublished observation). Login to comment
129 Changes in anion conduction brought about by titration of T338C CFTR are consistent with the predictions of a simplified charged-vestibule model We compared the effect of charge changes at 338 to the predictions of simplified charged-vestibule models used previously to analyze the influence of charge at position 334 (Smith et al., 2001). Login to comment
136 Single-channel conductance of T338C CFTR is pH-sensitive Fig. 5 A contains examples of T338C CFTR single-channel currents recorded from detached inside-out patches with the pH of the pipette solution (containing 1 mM 2-ME) buffered to either pH 6 or pH 7.4. Login to comment
143 We speculate that these events might be attributed to the opening of some endogenous channels or alternatively, to T338C CFTR channels that were in oxidation FIGURE 3 The pH-sensitivity of T338C CFTR conductance was due to the titration of the cysteine thiol. Login to comment
144 Oocytes expressing T338C or T338S CFTR were first exposed to a stimulatory cocktail containing 10 mM Isop and 1 mM IBMX at pH 7.4. Login to comment
148 Premodification by NEM prevented the large pH-induced response seen in T338C CFTR conductance. Login to comment
149 T338S CFTR exhibited no pH-sensitivity. Login to comment
156 It is also of interest to note that the ratio of the single channel conductance at pH 6 and pH 7.4 for T338C CFTR was ;1.8, a value comparable to the ratio observed for the macroscopic conductances (1.7). Login to comment
166 The macroscopic conductance due to T338C CFTR exhibits a pKa that is more acidic than expected for a simple thiol in free solution Shown in Fig. 7 is a representative titration curve obtained by measuring the conductance of an oocyte expressing T338C CFTR at different values of bath pH (n ¼ 5). Login to comment
169 (A) Using the continuum model, the I-V curve corresponding to pH 6 (open squares) obtained from an oocyte expressing T338C CFTR was first fitted by fixing Co at zero, resulting in a PCl of 3.51 3 10ÿ7 cm3 /s, a Ci of 42 mM, and a Ci of 20.9 mV. Login to comment
184 To investigate the effect of charge at position 334 on the titration behavior of T338C CFTR, we examined the conductance of oocytes expressing double mutants, T338C/R334A, T338C/R334E, and T338C/R334D CFTR. Login to comment
185 Shown in Fig. 8 A are representative titration curves for the conductance for T338C CFTR and two of these double mutants (n ¼ 5 each). Login to comment
186 Neutralizing the charge at 334 (R334A) resulted in a pKa that was more than one pH unit more basic (8.78 6 0.03, n ¼ 4) than that determined for T338C CFTR. Login to comment
187 The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n ¼ 4 and 8.96 6 0.08 for T338C/R334E CFTR, n ¼ 5). Login to comment
190 To test the generality of titration results obtained with T338C CFTR, we compared the titration behavior of T338H CFTR with that of a double mutant, T338H/R334C, in which it was possible to change the charge at position 334 by means of chemical modification. Login to comment
191 Summarized in Fig. 8 B are the results obtained from oocytes expressing either T338H or chemically modified T338H/R334C CFTR (n ¼ 3-4 for each mutant). Login to comment
197 (A) Examples of single-channel current records obtained from inside-out patches detached from oocytes expressing T338C CFTR. Login to comment
198 (B) The i-V plot for T338C CFTR. Login to comment
211 Rates of covalent modification of T338C CFTR by MTSET1 and MTSES2 are consistent with a model featuring a positive vestibule potential If, as suggested by the shift in the apparent pKa of a cysteine or a histidine at 338, this portion of the CFTR pore is characterized by a positive electrostatic potential, the potential should also affect the relative rates of covalent modification by MTSET1 and MTSESÿ (Stauffer and Karlin, 1994; Pascual and Karlin, 1998; Karlin and Akabas, 1998). Login to comment
212 Fig. 9 contains the results of experiments in which the time course of modification was measured at pH 7.4 for oocytes expressing either T338C CFTR or a double mutant FIGURE 6 Alteration of charge at the engineered cysteine at position 338 did not affect channel gating. Login to comment
216 FIGURE 7 Titration of conductance due to T338C CFTR. Login to comment
221 FIGURE 8 The pH-induced changes in the conductances of oocytes expressing T338C/R334A, T338C/R334E, T338H, or T338H/R334C CFTR. Login to comment
222 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
228 in which the arginine at 334 was replaced by aspartic acid (T338C/R334D CFTR). Login to comment
229 Note that covalent modification of T338C CFTR with either reagent decreased the macroscopic conductance. Login to comment
232 Single-channel recordings (see below) showed that modification of T338C CFTR by MTSET1 reduced single-channel conductance, as expected if the ethyl(trimethylammonium) moiety partially obstructs anion flow through the pore. Login to comment
233 The rate of modification of T338C CFTR by MTSESÿ exceeded that for MTSET1 , a result that is even more remarkable given the fact that the intrinsic rate of reaction of MTSESÿ with simple thiols is ;12-fold less than that for MTSET1 , due to the electrostatic interaction of the thiolate anion and the MTSESÿ during thiol-disulfide exchange (Karlin and Akabas, 1998). Login to comment
234 Introduction of negative charge at 334 (in T338C/R334D) reversed the relative reaction rates so that modification by MTSET1 was more rapid. Login to comment
236 The time constants for MTSET1 and MTSESÿ modification of T338C CFTR ([MTS] ¼ 25 mM) averaged 64.5 6 2.2 s (n ¼ 3) and 11.3 6 1.9 s (n ¼ 3), respectively. Login to comment
237 The time constants for MTSET1 and MTSESÿ modification of T338C/R334D CFTR ([MTS] ¼ 25 mM) averaged 39.8 6 15.8 s (n ¼ 3) and 641 6 27.7 s (n ¼ 3), respectively. Login to comment
238 Calculations based on a simple kinetic model for the ratio of the rates of reaction of MTSET1 and MTSESÿ , including a correction for the difference in the intrinsic rates of the MTS-thiolate reactions (see Discussion), suggested that charged reagents modifying T338C CFTR sensed an electrostatic potential that was ;54 mV positive with respect to the bath. Login to comment
240 Covalent modification of T338C CFTR by MTSET1 adds a positive charge, but also partially obstructs the pore The reduction of T338C CFTR macroscopic conductance by MTSET1 suggested that at this locus, the deposition of the ethyl(trimethylammonium) moiety might partially block the pore. Login to comment
241 Direct evidence for pore obstruction by MTSET1 was obtained by recording T338C CFTR single-channel currents before and after modification. Login to comment
247 The effective concentration of MTSET1 in the pipette likely varied because the half-life of MTSET1 is only ;10 min at room temperature according to Stauffer FIGURE 9 Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET1 (n ¼ 3) and MTSESÿ (n ¼ 3). Login to comment
249 (A) After activation, oocytes expressing T338C CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSESÿ (:). Login to comment
250 (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSESÿ (d). Login to comment
265 This result is consistent with the notion that the 0.2-pA event represented MTSET1 -modified T338C CFTR channels. Login to comment
266 Based on the single-channel amplitude observed under reducing conditions, it seems likely that the 0.2-pA events included a small contribution from a background channel or a terminal oxidation state of T338C CFTR; see Materials and Methods. Login to comment
267 The fact that the modification of the T338C CFTR channel was not always complete may reflect the acidic pH as well as some degradation of the MTSET1 . Login to comment
271 As an additional test of the effect of MTSET1 we examined changes in single-channel current amplitude in experiments in which the recording pipette was backfilled with MTSET1 to FIGURE 10 MTSET1 modification of T338C CFTR single-channel conductance. Login to comment
278 The results suggested a 75% decrease in single channel current at Vm ¼ ÿ100 mV at pH 6 due to MTSET1 -induced modification of T338C CFTR. Login to comment
289 If, as suggested by single-channel recordings, MTSET1 partially obstructs the pore of T338C CFTR, we reasoned that the net effect of MTSET1 on macroscopic conductance might consist of two components: one due to obstruction and another due to the change in charge. Login to comment
290 In previous experiments using R334C CFTR (Smith et al., 2001) we showed that, in general, the total charge change due to covalent modification was the sum of the charge added by the deposited group and the pH-dependent charge on the thiolate that is neutralized in the formation of a mixed disulfide bond. Login to comment
291 The results shown in Fig. 12 compare the effectsof covalent modificationof T338C CFTR at pH 6 and pH 9, values chosen such that the partial charge on the cysteine thiolate would vary from near zero (pH 6) to near ÿ1 (pH 9). Login to comment
292 At pH 9, MTSET1 increased T338C CFTR conductance by 50-110% (84 6 15, n ¼ 3) whereas at pH 6, the same treatment reduced the conductance due to T338C CFTR by 20-80% (n ¼ 16). Login to comment
295 This apparent twofold difference in efficacy is FIGURE 11 Real-time modification of T338C-CFTR channels activated by PKA and ATP in an excised, inside-out patch. Login to comment
297 During the course of the recording, MTSET1 diffused to the pipette tip, and modified the T338C-CFTR channels there. Login to comment
305 Cheung and Akabas (1996) reported that T338C CFTR did not react with externally applied MTSET1 or MTSESÿ and concluded that the residue did not lie on the outward-facing, water-accessible surface of the protein. Login to comment
313 The observation that modification of T338C CFTR by MTSET1 produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn. Login to comment
314 The pKa of T338C CFTR is consistent with a model in which T338C resides in the pore where it senses a positive electrostatic potential The pH titration of the conductances of oocytes expressing T338C CFTR was consistent with the stabilizing effect of a nearby positive charge on the thiolate anion, and the behavior of double mutants suggested that one source of this charge could be R334. Login to comment
318 FIGURE 12 The pH-dependent effect of MTSET1 on whole-cell T338C CFTR conductance. Login to comment
320 (A) At pH 6, MTSET1 reduced the conductance due to T338C CFTR by variable amount (20-80%). Login to comment
325 In the case of T338C CFTR, although we lack the information as to a three-dimensional structure, we can nevertheless make use of some of the general principles that have emerged from the analysis of other proteins. Login to comment
329 The total potential, CT o ; for T338C CFTR, would also contain a pH-dependent component due to the cysteine thiolate at 338. Login to comment
330 As indicated above, at alkaline pH, the charge at 338 would be likely to make a large contribution to CT o in T338C CFTR. Login to comment
338 The pKa values range from 8.3 for free cysteine to ;10.3 for methylthiol or ethylthiol, and would translate into values for Cq o ranging from 53 mV to 171 mV positive with respect to the bath using a pKa of 7.4 for T338C CFTR. Login to comment
340 A comparison of the apparent pKa of T338C CFTR with that of the double mutant, T338C/R334A, suggests that the amino acid substitution at position 334 shifted the pKa from ;7.4 to 8.8 or ;1.4 units. Login to comment
342 A comparison of T338C/R334A (pKa ¼ 8.8) with T338C/R334E (pKa ¼ 8.9), would suggest a further change in Cq o of ;ÿ6 mV. Login to comment
343 The relative rates of modification of T338C CFTR by MTSET1 and MTSESÿ provide another estimate of Cq o (Stauffer and Karlin, 1994; Pascual and Karlin, 1998; Karlin and Akabas, 1998; Wilson et al., 2000; Elinder et al., 2001). Login to comment
345 The ratio of the rates of modification is given by Eq. 7 (see Supplementary Material), k MTSES k MTSET ¼ k MTSES i k MTSET i exp 2 F RT Cq o : (7) Using the ratio for kMTSES i =kMTSET i of 1/12 measured for 2-ME (Karlin and Akabas, 1998), the relative rates of modification for these two compounds yields a value for Cq o of 54 mV for T338C CFTR and ÿ4 mV for T338C/R334D CFTR, comparable to the values estimated from the shift in cysteine pKa resulting from the replacement of arginine by alanine or aspartic acid. Login to comment
347 Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles. Login to comment
356 The results obtained using two rather different approaches are both consistent with the hypothesis that the observed shift in the apparent pKa of T338C CFTR could be the result of the electrostatic effect of R334. Login to comment
360 It would not be surprising to find that the thiolate- arginine ion pair of T338C/R334 was closer together than the thiolate-glutamic acid pair of T338C/R334E, even if there were no major change in structure that accompanied the mutations. Login to comment
54 In studies of T338C CFTR, the pipette solution contained 196 mM NMDG-Cl, 2 mM MgCl2, and 5 mM HEPES HemiNa, pH 7.4; 5 mM MES was used in place of HEPES when a pH of 6 was desired for the pipette solution. Login to comment
107 Fig. 2 A contains a plot of gCl at Vm &#bc; Erev versus time from a representative experiment (n &#bc; 5) designed to test the pH-sensitivity of T338C CFTR. Login to comment
114 It can be seen that as the bath pH became more acidic the I-V plot changed in two ways: gCl at Vm &#bc; Erev FIGURE 2 The pH-induced change in conductance of oocytes expressing T338C CFTR. Login to comment
127 As summarized in Fig. 3, acidifying the bathing solution (pH 7.4-6) induced an ;68% (612) increase in conductance in oocytes expressing T338C CFTR whereas alkalinizing the bath (pH 7.4-9) decreased the conductance by ;80% (64). Login to comment
130 Changes in anion conduction brought about by titration of T338C CFTR are consistent with the predictions of a simplified charged-vestibule model We compared the effect of charge changes at 338 to the predictions of simplified charged-vestibule models used previously to analyze the influence of charge at position 334 (Smith et al., 2001). Login to comment
137 Single-channel conductance of T338C CFTR is pH-sensitive Fig. 5 A contains examples of T338C CFTR single-channel currents recorded from detached inside-out patches with the pH of the pipette solution (containing 1 mM 2-ME) buffered to either pH 6 or pH 7.4. Login to comment
145 Oocytes expressing T338C or T338S CFTR were first exposed to a stimulatory cocktail containing 10 mM Isop and 1 mM IBMX at pH 7.4. Login to comment
157 It is also of interest to note that the ratio of the single channel conductance at pH 6 and pH 7.4 for T338C CFTR was ;1.8, a value comparable to the ratio observed for the macroscopic conductances (1.7). Login to comment
167 The macroscopic conductance due to T338C CFTR exhibits a pKa that is more acidic than expected for a simple thiol in free solution Shown in Fig. 7 is a representative titration curve obtained by measuring the conductance of an oocyte expressing T338C CFTR at different values of bath pH (n &#bc; 5). Login to comment
170 (A) Using the continuum model, the I-V curve corresponding to pH 6 (open squares) obtained from an oocyte expressing T338C CFTR was first fitted by fixing Co at zero, resulting in a PCl of 3.51 3 107 cm3 /s, a Ci of 42 mM, and a Ci of 20.9 mV. Login to comment
188 The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n &#bc; 4 and 8.96 6 0.08 for T338C/R334E CFTR, n &#bc; 5). Login to comment
199 (B) The i-V plot for T338C CFTR. Login to comment
213 Fig. 9 contains the results of experiments in which the time course of modification was measured at pH 7.4 for oocytes expressing either T338C CFTR or a double mutant FIGURE 6 Alteration of charge at the engineered cysteine at position 338 did not affect channel gating. Login to comment
217 FIGURE 7 Titration of conductance due to T338C CFTR. Login to comment
223 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
230 Note that covalent modification of T338C CFTR with either reagent decreased the macroscopic conductance. Login to comment
235 Introduction of negative charge at 334 (in T338C/R334D) reversed the relative reaction rates so that modification by MTSET1 was more rapid. Login to comment
239 Calculations based on a simple kinetic model for the ratio of the rates of reaction of MTSET1 and MTSES , including a correction for the difference in the intrinsic rates of the MTS-thiolate reactions (see Discussion), suggested that charged reagents modifying T338C CFTR sensed an electrostatic potential that was ;54 mV positive with respect to the bath. Login to comment
242 Direct evidence for pore obstruction by MTSET1 was obtained by recording T338C CFTR single-channel currents before and after modification. Login to comment
248 The effective concentration of MTSET1 in the pipette likely varied because the half-life of MTSET1 is only ;10 min at room temperature according to Stauffer FIGURE 9 Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET1 (n &#bc; 3) and MTSES (n &#bc; 3). Login to comment
251 (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSES (d). Login to comment
268 The fact that the modification of the T338C CFTR channel was not always complete may reflect the acidic pH as well as some degradation of the MTSET1 . Login to comment
272 As an additional test of the effect of MTSET1 we examined changes in single-channel current amplitude in experiments in which the recording pipette was backfilled with MTSET1 to FIGURE 10 MTSET1 modification of T338C CFTR single-channel conductance. Login to comment
279 The results suggested a 75% decrease in single channel current at Vm &#bc; 100 mV at pH 6 due to MTSET1 -induced modification of T338C CFTR. Login to comment
293 At pH 9, MTSET1 increased T338C CFTR conductance by 50-110% (84 6 15, n &#bc; 3) whereas at pH 6, the same treatment reduced the conductance due to T338C CFTR by 20-80% (n &#bc; 16). Login to comment
296 This apparent twofold difference in efficacy is FIGURE 11 Real-time modification of T338C-CFTR channels activated by PKA and ATP in an excised, inside-out patch. Login to comment
298 During the course of the recording, MTSET1 diffused to the pipette tip, and modified the T338C-CFTR channels there. Login to comment
307 Cheung and Akabas (1996) reported that T338C CFTR did not react with externally applied MTSET1 or MTSES and concluded that the residue did not lie on the outward-facing, water-accessible surface of the protein. Login to comment
315 The observation that modification of T338C CFTR by MTSET1 produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn. Login to comment
316 The pKa of T338C CFTR is consistent with a model in which T338C resides in the pore where it senses a positive electrostatic potential The pH titration of the conductances of oocytes expressing T338C CFTR was consistent with the stabilizing effect of a nearby positive charge on the thiolate anion, and the behavior of double mutants suggested that one source of this charge could be R334. Login to comment
322 (A) At pH 6, MTSET1 reduced the conductance due to T338C CFTR by variable amount (20-80%). Login to comment
327 In the case of T338C CFTR, although we lack the information as to a three-dimensional structure, we can nevertheless make use of some of the general principles that have emerged from the analysis of other proteins. Login to comment
331 The total potential, CT o ; for T338C CFTR, would also contain a pH-dependent component due to the cysteine thiolate at 338. Login to comment
332 As indicated above, at alkaline pH, the charge at 338 would be likely to make a large contribution to CT o in T338C CFTR. Login to comment
344 A comparison of T338C/R334A (pKa &#bc; 8.8) with T338C/R334E (pKa &#bc; 8.9), would suggest a further change in Cq o of ;6 mV. Login to comment
348 The absolute values of Cq o must be interpreted with caution since we do not know to what extent structural differences between these two mutants might have resulted from the amino acid substitution (see below), but both of the measurements used to estimate the electrostatic potential of the vestibule indicated an asymmetry between the impact of basic and acidic residues at 334. Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles. Login to comment
357 The results obtained using two rather different approaches are both consistent with the hypothesis that the observed shift in the apparent pKa of T338C CFTR could be the result of the electrostatic effect of R334. Login to comment
361 It would not be surprising to find that the thiolate- arginine ion pair of T338C/R334 was closer together than the thiolate-glutamic acid pair of T338C/R334E, even if there were no major change in structure that accompanied the mutations. Login to comment

[hide] Identification of cystic fibrosis transmembrane co... Biophys J. 1996 Jun;70(6):2688-95. Cheung M, Akabas MH
Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.
Biophys J. 1996 Jun;70(6):2688-95., [PMID:8744306]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
91 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
90 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment

[hide] CFTR: Ligand exchange between a permeant anion ([A... Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9. Serrano JR, Liu X, Borg ER, Alexander CS, Shaw CF 3rd, Dawson DC
CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore.
Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9., [PMID:16766608]

Abstract [show]
Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
0 CFTR: Ligand Exchange between a Permeant Anion ([Au(CN)2]2 ) and an Engineered Cysteine (T338C) Blocks the Pore Jose &#b4; R. Serrano,* Xuehong Liu,* Erik R. Borg,* Christopher S. Alexander,* C. Frank Shaw 3rd,y and David C. Dawson* *Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239; and y Department of Chemistry, Illinois State University, Normal, Illinois 61790-4160 ABSTRACT Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET1 and MTSES . Login to comment
2 Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2] produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). Login to comment
19 To test this hypothesis we used a CFTR construct with a cysteine substituted at position 338, T338C CFTR. Login to comment
20 We recently reported that the functional effects of both covalent and noncovalent modification of T338C CFTR were consistent with the hypothesis that a cysteine substituted at this location lies within the anion conduction path (8). Login to comment
36 Most importantly, the construct usedin the experiments reported here, T338C on a Cys-less background (T338C/Cys-less), behaved in a manner that was virtually identical to that of T338C/wt; including similar pH titration of macroscopic conductance (8). Login to comment
45 This variable reactivity is preserved in the T338C/Cys-less construct and cannot, therefore, be attributed to an intrapeptide disulfide bond. Login to comment
64 RESULTS T338C CFTR displays enhanced affinity for [Au(CN)2]2 We reported previously that wt CFTR is reversibly blocked by [Au(CN)2] (7), a result that was confirmed and extended to several CFTR mutants by Linsdell et al. (15-17). Login to comment
71 In the experiment depicted in Fig. 2 A, after exposure to DTT, exposure of T338C/wt to 100 mM [Au(CN)2] , a concentration of the pseudohalide that produced only modest inhibition of Cys-less CFTR conductance, virtually abolished the T338C/wt CFTR conductance. Login to comment
74 As indicated in Fig. 2 B, profound inhibition of conductance due to T338C/Cys-less CFTR that was not reversed by washing could be achieved with as little as 100 nM [Au(CN)2] , albeit at a slower rate. Login to comment
96 Fig. 2 C contains an example of an oocyte expressing T338C/Cys-less CFTR in which it was possible to observe high affinity, irreversible block, and lyotropic block by [Au(CN)2] . Login to comment
98 Over the course of these experiments we found that in oocytes pretreated with a reducing agent (2-ME or DTT) the extent of inhibition of T338C/(wt or Cys-less) FIGURE 2 [Au(CN)2] produced a profound, irreversible reduction of gCl in oocytes expressing T338C CFTR. Login to comment
120 This result suggests that the variable conductance that remains after irreversible block of T338C/Cys-less by micromolar concentrations of [Au(CN)2] represents CFTR channels that are not susceptible to high affinity block but remain susceptible to lyotropic block. Login to comment
122 This result is consistent with the hypothesis that those spontaneously reacting T338C CFTR channels in which the thiol cannot be recovered by exposure to 2-ME or DTT do not differ from wt CFTR as regards their susceptibility to lyotropic block. Login to comment
125 The results described above are consistent with the pattern of reactivity previously reported for T338C CFTR (13). Login to comment
126 T338C channels that have been previously exposed to 2-ME or DTT comprise at least two subpopulations. Login to comment
128 Exposure of these channels to nanomolar concentrations of [Au(CN)2] produces a profound, irreversible block that accounts for the majority of the inhibition of macroscopic conductance due to T338C/wt or T338C/Cys-less CFTR. Login to comment
136 An oocyte expressing T338C/wt CFTR was exposed to 1 mM 2-ME (open circles); 100 mM [Au(CN)2] (solid circles) irreversibly reduced gCl by ;80%. Login to comment
139 Exposure of an oocyte expressing T338C/wt CFTR to 100 mM [Au(CN)2] resulted in nearly 100% inhibition of the conductance. Login to comment
142 High affinity block of T338C CFTR by [Au(CN)2] was similarly abolished by exposing oocytes to either MTSET1 or MPA, a polar malemide (not shown). Login to comment
144 High affinity block of T338C CFTR conductance: evidence for a ligand-exchange reaction The irreversible block of T338C CFTR conductance by [Au(CN)2] suggests that the anion participates in a high affinity interaction with the substituted cysteine. Login to comment
147 If the reaction between [Au(CN)2] and T338C CFTR occurs via the mechanism depicted above then it should be possible to reverse the reaction by supplying excess cyanide ligand. Login to comment
149 After activating T338C/Cys-less CFTR and treating the oocyte with 2-ME, exposure to 0.5 mM KCN resulted in an 18% reduction of the conductance that was readily reversed by washing with a KCN-free solution (Fig. 5 A). Login to comment
165 Both anions produced only rapidly reversible, lyotropic block of T338C/Cys-less CFTR that was not affected by blocking the thiolate with IAM (not shown). Login to comment
167 6 and 7 compare the effects of [Au(CN)2] on Cys-less and T338C/Cys-less CFTR channels recorded from detached, inside out patches. Login to comment
176 This observation differs from that reported by Linsdell and Gong (17) for wt CFTR, suggesting that the interaction of [Au(CN)2] with endogenous cysteines not present in the Cys-less construct may account for the effect on open probability. Exposure to [Au(CN)2] produced a strikingly different response in T338C/Cys-less CFTR channels (n &#bc; 10). Login to comment
183 (A) Oocytes expressing T338C/Cys-less CFTR were activated and exposed first to 1 mM 2-ME (open circles) and then to 500 mM KCN (shaded triangles). Login to comment
187 After activation, oocytes expressing T338C/Cys-less CFTR were exposed to 1 mM 2-ME (open circles) and then to 1 mM [Au(CN)2] (solid circles). Login to comment
190 that the more rare, 0.4 pA events could represent a substate of the T338C/Cys-less channel (22). Login to comment
195 The behavior of this patch was consistent with the notion that it contained T338C/Cys-less CFTR channels that were blocked by [Au(CN)2] and a second channel that was unreactive, yet dependent upon PKA and ATP. Login to comment
198 DISCUSSION High affinity block of T338C CFTR by a permeant anion [Au(CN)2] is a very stable coordination compound with a log stability constant (log K) of 37-39 (20,21); where K is given by K &#bc; &#bd;Au&#f0;CN&#de;2 &#bd;Au 1 &#bd;CN 2: (2) Yet, in a solution containing excess [CN] , individual [CN] undergoes exchange at the Au(I) center at the fast exchange limit detected by 13 C-NMR (23). Login to comment
208 Mechanism of irreversible inhibition of CFTR conductance The profound, rapid decrease in the conductance of oocytes expressing T338C CFTR could reflect a decrease in single-channel conductance, a reduction of channel open probability, or some combination of these two effects. Login to comment
212 Taken together, these observations suggest that at least a portion of the decrease in single-channel conductance produced by exposure of T338C CFTR to [Au(CN)2] reflects the deposition of a negative charge within the pore via the mixed ligand complex, Protein-S-[Au-CN] . Login to comment
213 In FIGURE 7 [Au(CN)2] irreversibly blocked T338C/Cys-less CFTR single-channel currents. Login to comment
229 It is important to note, however, that the single-channel studies reported here were conducted using Cys-less CFTR and T338C/Cys-less CFTR to avoid confounding effects resulting from endogenous cysteines that are accessible to reagents applied to the cytoplasmic face of detached patches (10). Login to comment
231 In any single oocyte expressing T338C CFTR that has been previously exposed to 2-ME or DTT, the CFTR channels appear tocompriseamixedpopulation.In somechannelsthecysteineat 338 is in the simple thiolate form, whereas in others the thiol has undergone a chemical reaction that renders the thiolate unavailable for ligand exchange or thiol-disulfide exchange reactions. Login to comment

[hide] Cysteine scanning of CFTR's first transmembrane se... Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048. Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PMID:23442957]

Abstract [show]
Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
8 Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. Login to comment
128 As shown in Fig. S1 in the Supporting Material, the pKa value of C107 is 7.25, which is nearly identical to that of T338C in TM6 (42). Login to comment

[hide] Relative contribution of different transmembrane s... Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20. Wang W, El Hiani Y, Rubaiy HN, Linsdell P
Relative contribution of different transmembrane segments to the CFTR chloride channel pore.
Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20., [PMID:23955087]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
4 Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. Login to comment
127 State-dependent accessibility of T1115C and S1118C in TM11 Modification of T1115C and S1118C by both internal and external MTSES is reminiscent of the accessibility pattern observed for TM6 cysteine mutant T338C [42]. Login to comment
128 This similarity is perhaps not surprising since a disulfide bond can be formed between the two cysteine side chains of T338C and S1118C in open channels [43], indicating close physical proximity of these pore-exposed side chains. Login to comment
129 Previously we suggested that the ability of T338C to be modified by MTSES from both sides of the membrane was due to this residue Fig. 5 Single channel currents carried by TM11 mutant channels. Login to comment
141 This suggestion is consistent with data that S1118C can form a disulfide bond with T338C in open channels [43]. Login to comment
187 The proposed relative alignment of TMs 6 and 11 presented in Fig. 9a is consistent not only with the pattern of MTSES accessibility from different sides of the membrane, but also with previous data showing that disulfide bonds can form between T338C and S1118C, and between R334C and T1122C, in open channels [43]. Login to comment
199 Furthermore, using the same approach used previously to study side-dependent modification of T338C [42], we found that T1115C and S1118C were more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels (Fig. 4). Login to comment
200 This pattern, which is common to that previously observed for T338C [42], is consistent with these residues showing alternating access to the extracellular and intracellular sides of the membrane during channel opening and closing (Fig. 9c). Login to comment

[hide] Modeling the conformational changes underlying cha... PLoS One. 2013 Sep 27;8(9):e74574. doi: 10.1371/journal.pone.0074574. eCollection 2013. Rahman KS, Cui G, Harvey SC, McCarty NA
Modeling the conformational changes underlying channel opening in CFTR.
PLoS One. 2013 Sep 27;8(9):e74574. doi: 10.1371/journal.pone.0074574. eCollection 2013., [PMID:24086355]

Abstract [show]
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Changes in side-chain interactions are observed in all major domains of the protein, and experimental confirmation was obtained for a novel intra-protein salt bridge that breaks near the end of the transition. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
410 Serrano JR, Liu X, Borg ER, Alexander CS, Shaw CF, et al. (2006) CFTR: Ligand exchange between a permeant anion ([Au(CN)2]2 ) and an engineered cysteine (T338C) blocks the pore. Biophys J 91: 1737-1748. doi:10.1529/ biophysj.105.078899. Login to comment

[hide] Localizing a gate in CFTR. Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9. Gao X, Hwang TC
Localizing a gate in CFTR.
Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9., [PMID:25675504]

Abstract [show]
Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR's gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2](-), we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338-341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2](-) in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2](-) in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2](-) when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2](-) and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
78 State-Dependent Reactivity of T338C, F337C, and R334C Implicates the Location of a Gate for CFTR. Login to comment
84 As shown in Fig. 2A, the application of just 50 bc;M [Au(CN)2]- in the presence of ATP abolished over 90% of the T338C-CFTR current in an inside-out patch, and this reduction of the current is attributed to the stable coordination between 338C and the probe because removal of [Au(CN)2]- from the solution failed to recover the current. Login to comment
87 To the contrary, the macroscopic current of T338C-CFTR remained almost constant when [Au(CN)2]- was perfused to the patch in the absence of ATP (Fig. 2B). Login to comment
96 Our previous studies demonstrated that a disease-associated mutation G1349D could decrease the Po of CFTR by ~10-fold (34) without affecting trafficking of the channel (34, 35); we thus engineered this mutation into R334C, K335C, F337C, and T338C backgrounds. Login to comment
97 As shown in Fig. S5, indeed, introducing the G1349D mutation into T338C-CFTR lowered the Po and decreased the reaction rate by ~10-fold, which can be interpreted as a limited accessibility of the side chain of 338C in the closed state. Login to comment
114 State-dependent reactivity of T338C-CFTR to internal [Au(CN)2]- . Login to comment
115 (A) In the presence of ATP, internal [Au(CN)2]- irreversibly decreases T338C-CFTR currents. Login to comment
117 (B) Contrary to that seen in A, T338C-CFTR currents were not significantly altered when the same concentration [Au(CN)2]- was applied in the absence of ATP for a total of 36 s. considering the side chain of 344C is accessible by intracellular [Au(CN)2]- regardless of whether the channel is open or closed (Fig. 1 C and D), we propose a gate residing in between positions 337 and 344 along TM6 for CFTR (Fig. 1A). Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment
313 Serrano JR, et al. (2006) CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore. Biophys J 91(5):1737-1748. Login to comment