ABCMdb

PMID: 16436375

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 [PubMed]

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0 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338His 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys A, effect of 2-ME on T338C/WT CFTR conductance. Login to comment 57 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys A similar pattern of reactivity was observed in oocytes expressing T338C/Cys-less CFTR (Fig. 1C, inset). Login to comment 83 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 102 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ser This 6 Similarly, exposure to reducing agents was without effect on oocytes expressing either T338A or T338S CFTR, constructs that retain the 18 endogenous cysteines (see below). Login to comment 103 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 162 ABCC7 p.Thr338Ala C, oocytes expressing T338A CFTR were first exposed to: 1 mM Au(CN)2 - (black circles), 1 mM IAM, 1 mM Au(CN)2 - . Login to comment 164 ABCC7 p.Thr338Cys Different Chemical States of an Engineered Cysteine (T338C) 8280 with the hypothesis that channels reacting with IAM originated from two populations. Login to comment 167 ABCC7 p.Thr338Ala Fig. 4C contains the result of an experiment showing that IAM did not affect the function of T338A CFTR (n ϭ 3). Login to comment 169 ABCC7 p.Thr338Ala Block of T338A CFTR was unaffected by exposure to IAM. Login to comment 170 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 200 ABCC7 p.Thr338Ser Up to 14 mM H2O2 had no discernable effect on conductance of oocytes expressing T338S or WT CFTR.7 Low Concentrations of Copper Mimicked the gSX1 Phenotype-Metals like iron, zinc, and copper that react with free thiols are abundant in Xeno- FIGURE 6. Login to comment 206 ABCC7 p.Thr338Cys Neither SNAP nor H2O2 mimicked the phenotype of T338C CFTR. Login to comment 210 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys Exposure of copper-inhibited oocytes expressing T338C/WT to MTSETϩ mimicked the phenotype seen in naı¨ve oocytes. Login to comment 228 ABCC7 p.Thr338Cys Copper mimicked the phenotype seen in naı¨ve oocytes expressing T338C CFTR. Login to comment 233 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys It seems appropriate to view the action of 2-ME and DTT on T338C CFTRasareflectionoftheirabilitytoligandmetalsratherthantheiractivity as "reducing agents." Login to comment 266 ABCC7 p.Thr338Cys 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 ABCC7 p.Thr338Cys 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