ABCMdb

ABCC7 p.Thr338Ser

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,

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Publications
[hide] Relationship between anion binding and anion perme... J Physiol. 2001 Feb 15;531(Pt 1):51-66. Linsdell P
Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol. 2001 Feb 15;531(Pt 1):51-66., 2001-02-15 [PMID:11179391]

Abstract [show]
1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies.

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No. Sentence Comment
264 Optimization of Cl¦ conductance, rather than Cl¦ permeability, is likely to be of greater importance to the physiological function of CFTR and other Cl¦ channels, although in CFTR the mutations T338A and T338S increase Cl¦ conductance, apparently without adversely affecting other channel properties (Linsdell et al. 1998). Login to comment

[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.

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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

[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.

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No. Sentence 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
102 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
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
200 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

[hide] Non-pore lining amino acid side chains influence a... J Physiol. 1998 Oct 1;512 ( Pt 1):1-16. Linsdell P, Zheng SX, Hanrahan JW
Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines.
J Physiol. 1998 Oct 1;512 ( Pt 1):1-16., 1998-10-01 [PMID:9729613]

Abstract [show]
1. The effects of individually mutating two adjacent threonine residues in the sixth membrane-spanning region (TM6) of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel on permeation properties were examined using patch clamp recording from mammalian cell lines stably expressing human CFTR. 2. A number of mutations of T338 significantly affected the permeation properties of the channel. Increases and decreases in single channel conductance were observed for different mutants. Anion selectivity was strongly affected, with no two channel variants sharing the same selectivity sequence. Several mutations led to strong inward rectification of the macroscopic current-voltage relationship. The effects of these mutations on permeation properties were correlated with the size of the amino acid side chain substituted, rather than its chemical nature. 3. Most mutations of T339 resulted in a lack of functional channel expression and apparent misprocessing of the protein. One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations. 4. These results suggest an important role for T338 in controlling the permeation properties of the CFTR Cl- channel. It is suggested that mutation of this residue alters the interaction between permeating anions and the channel pore via an indirect effect on the orientation of the TM6 helix.

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No. Sentence Comment
93 Single CFTR channel currents in inside-out patches excised from CHO cells stably expressing wild-type, T338A or T338S are shown in Fig. 4A. Login to comment
97 Mean slope conductance was increased from 7·9 ± 0·1 pS (n = 18) for wild-type to 10·4 ± 0·1 pS (n = 9) for T338A and 11·3 ± 0·2 pS (n = 5) for T338S. Login to comment
99 Although the gating of T338A and T338S channels was not studied in detail, no striking differences from wild-type were noted. Login to comment
107 Unitary properties of T338A and T338S CFTR A, examples of single channel activity for wild-type, T338A and T338S, recorded at -50 mV. Login to comment
109 B and C, mean single channel current-voltage relationships for wild-type, T338A (B) and T338S (C). Login to comment
142 Permeability of intracellular anions in wild-type and mutant CFTR Cl¦ channels ------------------------------------------------------------ Anion WT T338A T338S T338N T338V T339V ------------------------------------------------------------ Thiocyanate 2·63 ± 0·13 (6) 5·85 ± 0·27 (4)* 4·80 ± 0·19 (3)* 8·72 ± 1·03 (4)* 1·92 ± 0·35 (4)* 3·28 ± 0·08 (4)* Nitrate 1·53 ± 0·04 (7) 2·04 ± 0·08 (3)* 1·82 ± 0·03 (4)* 4·22 ± 0·22 (3)* 6·84 ± 1·18 (7)* 1·61 ± 0·02 (3) Bromide 1·23 ± 0·03 (5) 1·74 ± 0·04 (3)* 1·47 ± 0·07 (3)* 1·66 ± 0·15 (3)* 1·04 ± 0·09 (5) 1·39 ± 0·06 (4)* Chloride 1·00 ± 0·01 (10) 1·00 ± 0·02 (11) 1·00 ± 0·02 (6) 1·00 ± 0·03 (10) 1·00 ± 0·04 (11) 1·00 ± 0·06 (10) Iodide 0·84 ± 0·03 (5) 2·09 ± 0·16 (5)* 1·76 ± 0·09 (3)* 1·03 ± 0·05 (3)* 0·79 ± 0·11 (3) 0·84 ± 0·02 (3) Perchlorate 0·25 ± 0·02 (6) 1·35 ± 0·08 (3)* 0·66 ± 0·06 (3)* 0·41 ± 0·03 (3)* 0·54 ± 0·00 (3)* 0·24 ± 0·01 (4) Benzoate 0·069 ± 0·006 (6) 0·17 ± 0·03 (4)* 0·091 ± 0·019 (3) 0·089 ± 0·015 (4) 0·15 ± 0·02 (4)* 0·097 ± 0·014 (4) Hexafluorophosphate < 0·019 (4) 0·53 ± 0·01 (3)* 0·31 ± 0·02 (3)* 0·68 ± 0·02 (3)* 0·39 ± 0·05 (3)* 0·051 ± 0·010 (4)* Fluoride 0·11 ± 0·01 (7) 0·12 ± 0·02 (4) 0·095 ± 0·012 (4) 0·11 ± 0·01 (4) 0·093 ± 0·009 (3) 0·17 ± 0·02 (4)* Formate 0·25 ± 0·01 (8) 0·45 ± 0·04 (3)* 0·43 ± 0·03 (3)* 0·35 ± 0·04 (4)* 0·22 ± 0·01 (3) 0·28 ± 0·02 (3) Acetate 0·090 ± 0·004 (8) 0·19 ± 0·01 (3)* 0·18 ± 0·01 (3)* 0·10 ± 0·02 (5) 0·11 ± 0·02 (3) 0·16 ± 0·01 (3)* Propanoate 0·14 ± 0·01 (3) 0·18 ± 0·02 (4) 0·098 ± 0·010 (4)* 0·077 ± 0·013 (3)* 0·13 ± 0·02 (3) - Pyruvate 0·10 ± 0·01 (5) 0·20 ± 0·01 (3)* 0·13 ± 0·02 (3) 0·075 ± 0·015 (3) 0·17 ± 0·03 (3)* - Methane sulphonate 0·077 ± 0·005 (5) 0·14 ± 0·02 (4)* 0·079 ± 0·014 (3) 0·038 ± 0·004 (3)* 0·088 ± 0·007 (3) - Glutamate 0·096 ± 0·008 (4) 0·082 ± 0·009 (3) 0·080 ± 0·008 (3) 0·060 ± 0·012 (5)* 0·11 ± 0·01 (3) - Isethionate 0·13 ± 0·01 (4) 0·11 ± 0·01 (3) 0·086 ± 0·012 (5)* 0·043 ± 0·007 (3)* 0·067 ± 0·005 (3)* - Gluconate 0·068 ± 0·004 (36) 0·10 ± 0·01 (3)* 0·060 ± 0·004 (3) 0·044 ± 0·004 (3) 0·077 ± 0·009 (3) 0·088 ± 0·021 (5) ------------------------------------------------------------ Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.g. Figs 7 and 10) as described in Methods. Login to comment
150 Note that all lyotropic anions showed the permeability sequence T338A > T338S > wild-type, again suggesting that the effects of these mutations on pore properties are correlated with the size of the amino acid side chain substituted. Login to comment
153 Pore diameter of T338 mutants Previously, we suggested that the double mutant channel, TT338,339AA, had an increased minimum functional pore diameter, based on its increased permeability to extracellular formate, acetate, propanoate and pyruvate ions (Linsdell et P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.18 -------------------------------------------------------------------------------------------- Table 2 ---------------------------------------------- Wild-type SCN¦ > NO×¦ > Br¦ > Cl¦ > I¦ > ClOÚ¦ > formate > F¦ > PFÜ¦ T338A SCN¦ > I¦ ü NO×¦ > Br¦ > ClOÚ¦ > Cl¦ > PFÜ¦ > formate > F¦ T338S SCN¦ > NO×¦ ü I¦ > Br¦ > Cl¦ > ClOÚ¦ > formate > PFÜ¦ > F¦ T338N SCN¦ > NO×¦ > Br¦ > I¦ = Cl¦ > PFÜ¦ > ClOÚ¦ > formate > F¦ T338V NO×¦ > SCN¦ > Br¦ = Cl¦ > I¦ > ClOÚ¦ > PFÜ¦ > formate > F¦ -------------------------------------------------------------------------------------------- Figure 7. Login to comment
164 In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0·528 nm (wild-type), 0·576 nm (T338A), 0·549 nm (T338S), 0·510 nm (T338N) and 0·540 nm (T338V). Login to comment
168 In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0·528 nm for wild-type, 0·576 nm for T338A, 0·549 nm for T338S, 0·510 nm for T338N and 0·540 nm for T338V. Anions examined (in order of increasing diameter) were: SCN¦, Cl¦, NO×¦, Br¦, I¦, ClOÚ¦, benzoate and PFÜ¦. Login to comment
171 In this case, fits by eqn (2) suggested minimum pore diameters of 0·535 nm (wild-type), 0·615 nm (T338A), 0·505 nm (T338S), 0·503 nm (T338N) and 0·530 nm (T338V). Login to comment
179 As with T338A and T338S, T339V showed apparently normal channel gating, with open probability being time and P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.110 Figure 9. Login to comment
181 In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0·535 nm for wild type, 0·615 nm for T338A, 0·505 nm for T338S, 0·503 nm for T338N and 0·530 nm for T338V. Anions examined (in order of increasing diameter) were: formate, acetate, propanoate, pyruvate, methane sulphonate and gluconate.) Login to comment
196 Conversely, the elevated conductance of T338A and T338S might be advantageous in gene or protein replacement therapies for Alteration of CFTR anion selectivityJ. Physiol. 512.1 11 Figure 10. Login to comment
205 Interestingly, both T338A (10·4 pS; Fig. 4B) and T338S (11·3 pS; Fig. 4C) have higher conductances than that we reported previously for TT338,339AA (9·9 pS; Linsdell et al. 1997b). Login to comment
206 Thus T338S has the highest conductance of any CFTR variant described to date. 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.

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No. Sentence 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
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
149 T338S CFTR exhibited no pH-sensitivity. 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
150 T338S CFTR exhibited no pH-sensitivity. Login to comment