ABCMdb

ABCC7 p.Ile344Ala

Predicted by SNAP2:	A: N (66%), C: N (66%), D: D (59%), E: N (57%), F: N (53%), G: D (53%), H: D (53%), K: N (57%), L: N (78%), M: N (82%), N: N (57%), P: N (53%), Q: N (61%), R: N (66%), S: N (72%), T: N (66%), V: N (82%), W: D (66%), Y: D (53%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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Publications
[hide] Molecular determinants of Au(CN)(2)(-) binding and... J Physiol. 2002 Apr 1;540(Pt 1):39-47. Gong X, Burbridge SM, Cowley EA, Linsdell P
Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.
J Physiol. 2002 Apr 1;540(Pt 1):39-47., 2002-04-01 [PMID:11927667]

Abstract [show]
Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)(2)(-) anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 microM Au(CN)(2)(-), a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Relative Au(CN)(2)(-) permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337-S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent.

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No. Sentence Comment
12 Channel block by 100 mM Au(CN)2 _ , a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Login to comment
78 Currents carried by the CFTR mutants K329A, K335A, T338A, S341A and I344A were also stimulated an average of 2_3-fold by PPi (Fig. 2B). Login to comment
87 Comparison between different channel variants at _100 mV reveals the sensitivity to this concentration of Au(CN)2 _ is R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A. Login to comment
124 The sequence of relative sensitivity to block by 100 mM Au(CN)2 _ at _100 mV (R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A) suggests that T338 normally makes the strongest contribution to Au(CN)2 _ binding within the pore, with nearby residues K335 and F337 also making large contributions. Login to comment

[hide] Differential contribution of TM6 and TM12 to the p... Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13. Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PMID:22160394]

Abstract [show]
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 muM Glyb and 200 muM Glip was 0.64 +/- 0.03 (n = 7) and 0.48 +/- 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 muM Tolb was 0.40 +/- 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

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Sentences [show]
No. Sentence Comment
119 The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left). Login to comment
120 The results were nearly identical for block by Glip (Fig. 3 middle) with one important distinction that significant effects were seen with mutations I344A (for Glyb) and V345A (for Glip), suggesting that these two highly related molecules share binding sites. Login to comment
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment

[hide] Biochemical implications of sequence comparisons o... Arch Biochem Biophys. 2002 May 15;401(2):215-22. Tan AL, Ong SA, Venkatesh B
Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator.
Arch Biochem Biophys. 2002 May 15;401(2):215-22., [PMID:12054472]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is both of medical significance in humans and of interest with regard to osmoregulation in aquatic organisms. CFTR is composed of five domains: two membrane-spanning domains, two nucleotide-binding domains, and a regulatory domain. Notwithstanding the plethora of information concerning the structure and function of CFTR, the biochemistry of many facets of CFTR are not completely understood. In this regard, we have performed a sequence alignment of representative vertebrate CFTR with the aim of generating hypotheses on the functional significance of conserved and variable residues. Postulates on function common to all organisms are: (i) Thr338 in the sixth transmembrane segment could have a function related to that of the pore-lining residue Lys335, and it is possible that Thr338 hydrogen bonds to Lys335, thus indirectly affecting anion permeability; (ii) the fragment (111)PDNKE could be an ion sensor; (iii) motifs in the two nucleotide-binding domains reflect differential ATP binding and hydrolysis; and (iv) an interaction in the R domain involving (765)RRQSVL and the C terminal end of the domain results in an inhibitory conformation. Major adaptations in fishes include variations in the postulated ion sensor (111)PDNKE, and the absence of a proline residue in the R domain with consequent higher chloride efflux.

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Sentences [show]
No. Sentence Comment
72 The mutation I344A afforded a modest alteration in the anion permeability [31]. Login to comment
73 A noteworthy observation is that whereas ClÀ > IÀ in the wild type, ClÀ % IÀ for I344A. Login to comment

[hide] Molecular determinants of anion selectivity in the... Biophys J. 2000 Jun;78(6):2973-82. Linsdell P, Evagelidis A, Hanrahan JW
Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
Biophys J. 2000 Jun;78(6):2973-82., [PMID:10827976]

Abstract [show]
Ionic selectivity in many cation channels is achieved over a short region of the pore known as the selectivity filter, the molecular determinants of which have been identified in Ca(2+), Na(+), and K(+) channels. However, a filter controlling selectivity among different anions has not previously been identified in any Cl(-) channel. In fact, because Cl(-) channels are only weakly selective among small anions, and because their selectivity has proved so resistant to site-directed mutagenesis, the very existence of a discrete anion selectivity filter has been called into question. Here we show that mutation of a putative pore-lining phenylalanine residue, F337, in the sixth membrane-spanning region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, dramatically alters the relative permeabilities of different anions in the channel. Specifically, mutations that reduce the size of the amino acid side chain present at this position virtually abolish the relationship between anion permeability and hydration energy, a relationship that characterizes the anion selectivity not only of wild-type CFTR, but of most classes of Cl(-) channels. These results suggest that the pore of CFTR may indeed contain a specialized region, analogous to the selectivity filter of cation channels, at which discrimination between different permeant anions takes place. Because F337 is adjacent to another amino acid residue, T338, which also affects anion selectivity in CFTR, we suggest that selectivity is predominantly determined over a physically discrete region of the pore located near these important residues.

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Sentences [show]
No. Sentence Comment
70 The mutants F337L, F337Y, and I344A gave only modest alterations in anion permeability (Table 1) that led to only slight changes in the anion selectivity sequence (Table 2). Login to comment
77 Both wild-type and F337Y (Fig. 3), as well as F337L, F337W, and I344A (not shown; see Table 2), were able to select for anions that bound water molecules less strongly, consistent with the lyotropic selectivity sequence common to most classes of Clafa; channels (see Introduction). Login to comment
116 Although we cannot rule out this possibility, we feel that the fact that mutations at two adjacent TM6 residues, F337 (this study) and T338 (Linsdell et al., 1998), significantly affect TABLE 1 Relative permeability of intracellular ions in wild-type and mutant CFTR Cld1a; channels Wild type F337A F337S F337L F337Y F337W I344A Cl 1.00 afe; 0.01 (10) 1.00 afe; 0.04 (6) 1.00 afe; 0.08 (3) 1.00 afe; 0.02 (5) 1.00 afe; 0.02 (6) 1.00 afe; 0.03 (5) 1.00 afe; 0.01 (9) Br 1.37 afe; 0.07 (8) 0.60 afe; 0.04 (4)** 0.50 afe; 0.04 (4)** 1.22 afe; 0.04 (5) 1.39 afe; 0.04 (3) 1.12 afe; 0.05 (4)* 1.74 afe; 0.01 (3)* I 0.83 afe; 0.03 (6) 0.23 afe; 0.04 (5)** 0.23 afe; 0.02 (4)** 0.39 afe; 0.01 (3)** 0.69 afe; 0.03 (7)* - 0.99 afe; 0.05 (4)* F 0.103 afe; 0.007 (9) 0.35 afe; 0.01 (4)** 0.43 afe; 0.02 (4)** 0.15 afe; 0.02 (3)* 0.095 afe; 0.009 (3) 0.081 afe; 0.009 (3) 0.075 afe; 0.012 (5)* SCN 3.55 afe; 0.26 (7) 0.97 afe; 0.05 (4)** 0.93 afe; 0.10 (5)** 2.85 afe; 0.20 (4) 3.05 afe; 0.29 (4) 4.42 afe; 0.56 (4) 3.27 afe; 0.30 (5) NO3 1.58 afe; 0.04 (10) 1.30 afe; 0.03 (3)* 1.08 afe; 0.02 (4)** 1.38 afe; 0.03 (4)* 1.43 afe; 0.04 (3) 1.62 afe; 0.03 (3) 1.71 afe; 0.06 (4) ClO4 0.25 afe; 0.01 (8) 0.19 afe; 0.00 (3)* 0.17 afe; 0.03 (4)* 0.23 afe; 0.04 (3) 0.15 afe; 0.01 (4)** - 0.24 afe; 0.02 (3) Formate 0.24 afe; 0.01 (9) 0.27 afe; 0.02 (3) 0.33 afe; 0.03 (4)* 0.35 afe; 0.02 (3)* 0.24 afe; 0.01 (3) - 0.28 afe; 0.01 (3) Acetate 0.091 afe; 0.003 (10) 0.073 afe; 0.004 (3)* 0.12 afe; 0.02 (5) - 0.092 afe; 0.014 (4) - 0.076 afe; 0.007 (3) Naaf9; 0.007 afe; 0.010 (24) 0.001 afe; 0.018 (3) 0.001 afe; 0.021 (5) - 0.002 afe; 0.004 (3) - - Relative permeabilities for different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.g., Fig. 2), according to Eq. 1 (see Materials and Methods). Login to comment
122 TABLE 2 Anion selectivity sequences for wild-type and mutant CFTR Cld1a; channels Wild-type SCNafa; b0e; NO3 afa; b0e; Brafa; b0e; Clafa; b0e; Iafa; b0e; ClO4 afa; b07; form b0e; Fafa; b0e; ace F337A NO3 afa; b0e; Clafa; c56; SCNafa; b0e; Brafa; b0e; Fafa; b0e; form c56; Iafa; b0e; ClO4 afa; b0e; ace F337S NO3 afa; b0e; Clafa; c56; SCNafa; b0e; Brafa; b0e; Fafa; b0e; form b0e; Iafa; b0e; ClO4 afa; b0e; ace F337L SCNafa; b0e; NO3 afa; b0e; Brafa; b0e; Clafa; b0e; Iafa; b0e; form b0e; ClO4 afa; b0e; Fafa; F337Y SCNafa; b0e; NO3 afa; c56; Brafa; b0e; Clafa; b0e; Iafa; b0e; form b0e; ClO4 afa; b0e; Fafa; b07; ace I344A SCNafa; b0e; Brafa; c56; NO3 afa; b0e; Clafa; b07; Iafa; b0e; form b0e; ClO4 afa; b0e; ace b07; Fafa; Sequences were derived from the relative anion permeabilities given in Table 1. form, formate; ace, acetate. Login to comment
124 A mutation in TM6 that greatly reduced the size of a more distant putative pore-lining hydrophobic amino acid residue, I344A, had no strong effect on selectivity (Tables 1 and 2). Login to comment
158 While the mutants F337L, F337Y, and I344A maintain Eisenman sequence III, both F337A and F337S convert the channel to a relatively strong field strength sequence (Clafa; b0e; Brafa; b0e; Fafa; b0e; Iafa; ; Eisenman sequence V) (Table 2). Login to comment