ABCMdb

ABCC7 p.Ser1141Ala

Predicted by SNAP2:	A: N (53%), C: N (57%), D: D (66%), E: D (75%), F: D (71%), G: N (93%), H: D (75%), I: D (66%), K: D (80%), L: D (71%), M: D (59%), N: N (66%), P: D (75%), Q: D (53%), R: D (75%), T: D (53%), V: D (66%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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376 Gupta et al. [178] have examined the effect of T1134A, M1137A, N1138A, S1141A and T1142A and found that, in contrast to those in TM6, mutations in TM12 have little effect on channel permeation properties, suggesting that TM6 and TM12 make highly asymmetric contributions to the pore. Login to comment

[hide] Asymmetric structure of the cystic fibrosis transm... Biochemistry. 2001 Jun 5;40(22):6620-7. Gupta J, Evagelidis A, Hanrahan JW, Linsdell P
Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.
Biochemistry. 2001 Jun 5;40(22):6620-7., 2001-06-05 [PMID:11380256]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore.

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No. Sentence Comment
79 Five alanine-substitution mutations in TM12 were constructed: T1134A, M1137A, N1138A, S1141A, and T1142A (Figure 1B). Login to comment
81 Expression of these same mutants in CHO cells yielded similar results (data not shown), with the exception that S1141A expression could not be detected. Login to comment
92 Block of N1138A (Figure 2B), as well as T1134A, M1137A, and T1142A (data not shown), appeared somewhat weaker than for wild-type, whereas block of S1141A actually appeared stronger than for wild-type (Figure 2B). Login to comment
96 As described above, S1141A could not be expressed in CHO cells; however, unitary S1141A-CFTR Table 1: Relative Anion Permeabilities for Wild-Type and Mutant CFTRa wild-type T1134A M1137A N1138A S1141A T1142A Cl 1.00 ( 0.01 (10) 1.00 ( 0.06 (5) 1.00 ( 0.03 (6) 1.00 ( 0.02 (4) 1.00 ( 0.02 (5) 1.00 ( 0.04 (7) Br 1.37 ( 0.07 (8) 1.42 ( 0.03 (4) 1.61 ( 0.02 (5)* 1.32 ( 0.08 (7) 1.54 ( 0.05 (4) 1.44 ( 0.04 (5) I 0.83 ( 0.03 (6) 0.85 ( 0.04 (4) 0.88 ( 0.02 (3) 0.83 ( 0.03 (4) 0.78 ( 0.01 (3) 0.86 ( 0.03 (3) F 0.103 ( 0.007 (9) 0.077 ( 0.008 (3) 0.107 ( 0.014 (3) 0.089 ( 0.005 (4) 0.053 ( 0.003 (7)** 0.094 ( 0.007 (3) SCN 3.55 ( 0.26 (7) 3.70 ( 0.33 (4) 3.58 ( 0.14 (5) 3.53 ( 0.21 (6) 3.37 ( 0.35 (3) 3.64 ( 0.22 (5) NO3 1.58 ( 0.04 (10) 1.62 ( 0.05 (4) 1.60 ( 0.04 (3) 1.58 ( 0.05 (6) 1.57 ( 0.03 (3) 1.64 ( 0.07 (3) ClO4 0.25 ( 0.01 (8) 0.22 ( 0.00 (3) 0.29 ( 0.02 (3) 0.44 ( 0.05 (5)** 0.22 ( 0.01 (3) 0.30 ( 0.03 (5) formate 0.24 ( 0.01 (9) 0.26 ( 0.01 (3) 0.27 ( 0.03 (3) 0.27 ( 0.01 (4) 0.26 ( 0.02 (4) 0.25 ( 0.03 (3) acetate 0.091 ( 0.003 (10) 0.102 ( 0.021 (3) 0.103 ( 0.011 (3) 0.087 ( 0.022 (3) 0.066 ( 0.007 (4)* 0.086 ( 0.009 (3) a Relative permeabilities (PX/PCl) for different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials according to eq 1 (see Experimental Procedures), as described in detail previously (16, 20, 36). Login to comment
107 (B) Mean current remaining following addition of 10 mM SCN- (I/I0) for wild-type (O), N1138A (b, left panel) and S1141A (b, right panel), shown as a function of voltage. Login to comment
109 Block by SCN- appeared somewhat weakened in N1138A (left), as well as in T1134A, M1137A and T1142A (data not shown, but see Figure 5B), and strengthened in S1141A (right). Login to comment
112 All unitary currents were recorded in CHO cell patches, except S1141A, which was recorded in BHK cell patches. Login to comment
120 Because of the differences in CFTR channel gating between BHK and CHO cell membrane patches, the PO of S1141A was not measured. Login to comment
128 However, the degree of block was significantly reduced in T1134A, M1137A, and S1141A compared to wild-type (Figure 5). Login to comment
136 (A) Unitary currents carried by wild-type and S1141A-CFTR at -50 mV, with symmetrical 154 mM Cl-containing solutions (control) or after addition of 10 mM NaSCN to the intracellular solution. Login to comment
144 However, for N1138A, S1141A, and T1142A, different degrees of inhibition were observed, suggesting that SCN- has some other effect on these mutants which is both distinct from the observed reduction in unitary current amplitude and absent in wild-type CFTR. Since the single channel results report the isolated effects of SCN- on unitary current amplitude, which is presumably determined by the relative tightness of SCN- binding within the pore, we believe that the reduced apparent SCN- binding affinity suggested by the single channel results with T1134A, M1137A, and S1141A is the best reporter of altered pore function in these mutants. Login to comment
174 Block of unitary Cl- currents by SCN- was significantly weakened in T1134A, M1137A, and S1141A compared to wild-type (Figure 5). Login to comment

[hide] Point mutations in the pore region directly or ind... Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8. Gupta J, Linsdell P
Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.
Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8., [PMID:11889571]

Abstract [show]
The sulfonylurea glibenclamide is a relatively potent inhibitor of the CFTR Cl(-) channel. This inhibition is thought to be via an open channel block mechanism. However, nothing is known about the physical nature of the glibenclamide-binding site on CFTR. Here we show that mutations in the pore-forming 6th and 12th transmembrane regions of CFTR affect block by intracellular glibenclamide, confirming previous suggestions that glibenclamide enters the pore in order to block the channel. Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. These two mutations also abolished the dependence of block on the extracellular Cl(-) concentration, which in wild-type CFTR suggests an interaction between Cl(-) and glibenclamide within the channel pore that limits block. We suggest that mutations in the 12th transmembrane region strengthen glibenclamide block not by directly altering interactions between glibenclamide and the pore walls, but indirectly by reducing interactions between Cl(-) ions and glibenclamide within the pore. This work demonstrates that glibenclamide binds within the CFTR channel pore and begins to define its intrapore binding site.

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No. Sentence Comment
63 While block of the TM12 mutants S1141A (Fig. 1) and T1134A and M1137A (data not shown) was indistinguishable from wild-type, block was significantly weakened in the TM6 mutants F337A and T338A, and significantly strengthened in the TM12 mutants N1138A and T1142A (Fig. 1). Login to comment
71 In contrast, I/I0 was not altered in T1134A, M1137A or S1141A at any voltage (data not shown). Login to comment
116 This does not appear to be a nonspecific effect of mutagenesis within TM12, since three other mutations in this region (T1134A, M1137A, S1141A) had no effect on glibenclamide block (Fig. 3). Login to comment
135 Interestingly, these two mutations did not affect SCN- block, although other TM12 mutants (T1134A, M1137A, S1141A) did [10], suggesting that Cl- and SCN- binding may be controlled by different structural features within TM12. Login to comment

[hide] CFTR: mechanism of anion conduction. Physiol Rev. 1999 Jan;79(1 Suppl):S47-75. Dawson DC, Smith SS, Mansoura MK
CFTR: mechanism of anion conduction.
Physiol Rev. 1999 Jan;79(1 Suppl):S47-75., [PMID:9922376]

Abstract [show]
CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.

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576 An analogous tions with the peptide backbone of this model system might produce energy minima that would behave as bind-substitution in TM12 (S1141A) did not alter anion conduction or DPC block but, if residues surrounding S1141 were ing sites. 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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163 Effects on time-dependent block by mutations R334A and K335A Fractional block by Glip200 μM V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 * ** ** ** ** ** ** * V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 1.0 * * * * * ** ** ** ** Fractional block by Glyb50 μM Fig. 4 Alanine-scanning in TM12 to identify amino acids that interact with Glyb and Glip. Login to comment
192 S1141A (-0.83±0.02 pA, n=5) increased and T1134A (-0.59±0.02 pA, n=4) decreased single-channel full open state amplitude compared to WT-CFTR (-0.70±0.03 pA, n=10; Fig. 9). Login to comment
239 Hence, strong time-dependent block of macropatch currents, and the appearance of multiple drug-induced closed states in single-channel recordings, may not arise from 0.4 pA 2 s M348A c f 0.2 pA 2 s F337A c f 0.4 pA 2 s K335A c f 0.4 pA 2 s c s2 f D1152A 0.4 pA 2 s T1134A c f 0.4 pA 2 s S1141A c f s2 0.4 pA 2 s c f WT 2000 4000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 3000 9000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 6000 400 1200 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 800 1600 1000 3000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 2000 500 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 1000 4000 12000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 8000 200 600 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 400 Fig. 9 Representative single-channel traces for WT-, K335A-, F337A-, M348A-, T1134A-, S1141A-, and D1152A-CFTR (left) from excised inside-out membrane patches with symmetrical 150 mM Cl- solution, and their all-points amplitude histograms (right). Login to comment

[hide] Novel pore-lining residues in CFTR that govern per... Neuron. 1994 Sep;13(3):623-34. McDonough S, Davidson N, Lester HA, McCarty NA
Novel pore-lining residues in CFTR that govern permeation and open-channel block.
Neuron. 1994 Sep;13(3):623-34., [PMID:7522483]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is both a member of the ATP-binding cassette superfamily and a Cl(-)-selective ion channel. We investigated the permeation pathway of human CFTR with measurements on conduction and open-channel blockade by diphenylamine-2-carboxylic acid (DPC). We used site-directed mutagenesis and oocyte expression to locate residues in transmembrane domain (TM) 6 and TM 12 that contact DPC and control rectification and single-channel conductances. Thus, TM 12 and the previously investigated TM 6 line the CFTR pore. In each TM, residues in contact with DPC are separated by two turns of an alpha helix. The contributions of TM 6 and TM 12 to DPC block and Cl- permeation, however, are not equivalent. The resulting structural model for the conduction pathway may guide future studies of permeation in other Cl- channels and ATP-binding cassette transporters.

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No. Sentence Comment
61 The DPC-Binding Site Can Be Moved to TM 12 S1141 in TM 12 had a predicted position analogous to that of S341 in TM 6 (Figure I), yet mutation S1141A I (/AA) Figure 3. Login to comment
78 Affinity and Voltage Dependence for Block of CFTR Variants by DPC Construct TM Ko( - 100) (PM) 0 I-V Relation n Properties Wild type Wild type low [Cl-], (10 mM) K335E 6 K335F 6 T338A 6 T339A 6 S341A 6 S341T 6 S1118A 11 T1134A 12 T1134F 12 S1141A 12 Triple 6,12 276 f 14 181 f 13" 303 -t 14 351 * 15' 220 * 14 284 * 47 1251 f 116a 530 f 80" 243 * 37 230 * 20 74 * 3" 220 * 13 325 * 26b 0.41 f 0.01 0.32 f 0.02" 0.42 f 0.01 0.42 f 0.02 0.36 f 0.02" 0.44 * 0.12 0.49 * 0.03" 0.35 f 0.09 0.40 f 0.02 0.35 * 0.02" 0.41 f 0.01 0.42 f 0.03 0.21 * O.Ol",b Linear, E,,, = -8 f 1 mV Ere\ = +48+2mV Inward rectification Linear Linear Linear Strong inward rectification Inward rectification Linear Linear Linear Linear Strong inward rectification Affinity for DPC was determined empirically at -100 mV, from whole-cell currents measured in the presence of 200 uM DPC (see Experimental Procedures). Login to comment

[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]

Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

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20 Infrequent subconductance behavior is seen in some CFTR mutants, such as T338A/Cand S1141A-CFTR (7, 12). Login to comment