ABCMdb

ABCC7 p.Thr339Val

Predicted by SNAP2:	A: N (57%), C: D (75%), D: D (85%), E: D (91%), F: D (85%), G: D (80%), H: D (71%), I: D (71%), K: D (91%), L: D (75%), M: D (85%), N: D (75%), P: D (85%), Q: D (75%), R: D (91%), S: N (57%), V: D (63%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: D, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, V: N, W: D, Y: D,

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Publications
[hide] Identification of a region of strong discriminatio... Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67. McCarty NA, Zhang ZR
Identification of a region of strong discrimination in the pore of CFTR.
Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67., [PMID:11557589]

Abstract [show]
The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

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No. Sentence Comment
397 T339V CFTR, which did express in CHO cells, exhibited limited alterations in selectivity compared with mutants at T338 (24), consistent with a non-pore lining role for T339. Login to comment

[hide] Extent of the selectivity filter conferred by the ... Mol Membr Biol. 2003 Jan-Mar;20(1):45-52. Gupta J, Lindsell P
Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore.
Mol Membr Biol. 2003 Jan-Mar;20(1):45-52., [PMID:12745925]

Abstract [show]
Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl(-) channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.

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No. Sentence Comment
41 Example leak-subtracted I Á/V relationships obtained with different intracellular anions are shown for wild-type, R334C, F337A, T338A, T339V and S341A in Figure 2. Login to comment
43 Other mutants studied (K335A, I336A, I340A), like T339V, had only minor effects (data not shown). Login to comment
44 Of eight mutants studied, only T339V was without any significant effect on anion permeability (Table 1), and five mutations (R334C, K335A, F337A, T338A, I340A) led to changes in the permeability sequence among halides (Figure 2 and Table 2). Login to comment
59 Wild type R334C K335A I336A F337A T338A T339V I340A S341A Cl 1.009/0.00 (6) 1.009/0.01 (6) 1.009/0.05 (5) 1.009/0.01 (5) 1.009/0.02 (6) 1.009/0.02 (8) 1.009/0.03 (6) 1.009/0.02 (5) 1.009/0.01 (6) Br 1.479/0.06 (6) 0.969/0.00 (5)** 1.529/0.03 (5) 1.359/0.05 (5) 0.669/0.03 (6)** 2.209/0.05 (5)** 1.829/0.24 (5) 1.409/0.09 (6) 2.459/0.20 (5)** I 0.819/0.04 (6) 0.729/0.05 (3) 1.579/0.06 (4)** 0.589/0.02 (4)* 0.389/0.15 (3)* 2.799/0.26 (7)** 0.769/0.02 (6) 1.249/0.07 (6)** 0.739/0.06 (6) F 0.119/0.01 (6) 0.099/0.01 (3) 0.139/0.02 (3) 0.079/0.01 (5) 0.409/0.02 (4)** 0.139/0.01 (6) 0.079/0.00 (5) 0.069/0.01 (5) 0.059/0.01 (6)* SCN 4.759/0.30 (6) 2.769/0.38 (6)** 3.989/0.16 (5) 3.709/0.11 (5)* 1.269/0.12 (5)** 7.509/0.29 (6)** 4.829/0.40 (5) 4.189/0.14 (7)* 10.09/1.8 (6)* Relative permeabilities for different anions present in the intracellular solution under bi-ionic conditions were calculated from macroscopic current reversal potentials according to Eq. (1) (see Experimental procedures). Login to comment
65 Wild-type R334C K335A I336A F337A T338A T339V I340A S341A Cl (G(50/G'50) 1.039/0.09 (6) 4.509/0.60 (6)** 1.399/0.09 (5)** 1.519/0.14 (5)* 1.189/0.22 (6) 1.779/0.25 (8)* 1.199/0.06 (7)* 1.419/0.11 (5)* 1.809/0.18 (5)** Cl (GCl/GCl) 1.009/0.08 (6) 1.009/0.13 (6) 1.009/0.07 (5) 1.009/0.09 (5) 1.009/0.22 (6) 1.009/0.14 (8) 1.009/0.06 (7) 1.009/0.09 (5) 1.009/0.10 (5) Br 0.649/0.05 (6) 0.329/0.02 (6)** 0.669/0.05 (5) 1.079/0.10 (5)* 0.359/0.06 (6)** 0.499/0.03 (5) 0.659/0.09 (5) 0.669/0.08 (6) 1.529/0.30 (4)* I 0.299/0.05 (6) 0.749/0.02 (3)* 0.279/0.01 (4) 0.109/0.02 (4)* 0.349/0.08 (3) 0.389/0.03 (5) 0.309/0.05 (7) 0.279/0.03 (6) 1.049/0.16 (7)** F 0.379/0.04 (6) 0.329/0.04 (3) 0.349/0.03 (3) 0.709/0.10 (4)* 0.129/0.02 (3)* 0.239/0.02 (6)* 0.509/0.10 (4) 0.309/0.02 (5) 0.519/0.07 (6) SCN 0.389/0.02 (6) 0.339/0.03 (6) 0.669/0.10 (5)* 0.279/0.02 (6)* 0.399/0.04 (5) 0.269/0.02 (5)* 0.269/0.02 (4)* 0.359/0.04 (6) 0.839/0.14 (6)* Relative conductances for different anions were calculated from the slope of the macroscopic I Á/V relationship for inward versus outward currents (see Experimental procedures). Login to comment
86 Halide permeability sequence Eisenman sequence CFTR variants I( !/Br( !/Cl( !/F( I K335A, T338A Br( !/I( !/Cl( !/F( II I340A Br( !/Cl( !/I( !/F( III wild-type, I336A, T339V, S341A Cl( !/Br( !/I( !/F( IV R334C Cl( !/Br( !/F( !/I( V F337A Sequences were derived from the relative permeabilities given in table 1. Login to comment
124 In most (six of eight) cases, alanine substitution was employed; however, we have previously found that the mutants R334A [15] and T339A [22] fail to express in BHK cells, and for these residues mutants which gave adequate current expression (R334C, T339V) were studied. 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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Sentences [show]
No. Sentence Comment
20 One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations. Login to comment
60 In contrast, of five mutations made at T339, only one (T339V) produced detectable levels of CFTR protein in Western blots using cell lysates from one confluent 10 cm culture plate (approximately 2 ² 10É-3 ² 10É cells; Fig. 1D). 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
173 Effects of mutations at T339 Of five amino acid substitutions carried out at position 339, only one (T339V) resulted in the production of detectable amounts of CFTR protein (Fig. 1D). Login to comment
175 PKAand ATP-dependent currents were observed, however, in twelve of fourteen T339V CFTR patches under the same conditions (Fig. 10A). Login to comment
176 T339V showed slight inward current rectification under these symmetrical ionic conditions (-I+50ÏI-50 = 0·86 ± 0·01; n = 8). Login to comment
178 Mean chord conductances for T339V were 7·95 ± 0·16 pS (n = 4) at -50 mV and 6·37 ± 0·29 pS (n = 3) at +50 mV, compared with wild-type values of 7·91 ± 0·09 pS (n = 18) at -50 mV and 7·85 ± 0·09 pS (n = 12) at +50 mV. 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
183 Furthermore, as with T338 mutants, T339V had negligible cation permeability (data not shown). Login to comment
184 The T339V mutant also had significant alterations in ionic permeability (Fig. 10B and Table 1), although in general these were not as strong as those observed for T338 mutants. Login to comment
185 Indeed, the selectivity sequence for T339V (SCN¦ > NO×¦ > Br¦ > Cl¦ > I¦ > ClOÚ¦ > formate > F¦ > PFÜ¦) was the same as for wild-type (see Table 2). Login to comment
186 Gluconate permeability was not significantly altered in T339V, again suggesting no severe disruption of large organic anion permeability. Login to comment
187 All of these effects are consistent with the T339V mutant having less severely altered pore properties than any of the T338 mutants studied. Login to comment
197 Macroscopic current-voltage relationships for T339V CFTR A, T339V shows slight inward rectification with symmetrical Cl¦-containing solutions. Login to comment
198 B, example current-voltage relationships used to investigate the anion selectivity of T339V, measured under biionic conditions with ClOÚ¦ or NO×¦ in the intracellular solution. Login to comment
199 Note that the permeability of these ions in T339V is similar to that observed in wild-type (Fig. 7). Login to comment
209 Inward rectification is observed in several other TM6 mutants at the single channel level, e.g. K335E (Tabcharani et al. 1993), T339V (Fig. 11B) and I332K (P. Linsdell, J. A. Tabcharani & J. W. Hanrahan, unpublished observations); however, voltage-dependent gating in T338 mutants cannot be excluded. Login to comment
222 Unitary properties of T339V CFTR A, activity of a single T339V channel at -50 mV (cf. Login to comment
224 B, mean single channel current-voltage relationships for wild-type (as in Fig. 4B and C) and T339V. Login to comment
237 However, it is unclear why mutations at T339 should apparently be more sensitive to misprocessing than those at T338 (Fig. 1C and D), or why the non-conservative T339V mutation alone should be appropriately processed (Fig. 1D). Login to comment
240 Because of low protein expression, we were only able to characterize the permeation properties of one T339 mutant, T339V. Login to comment