ABCMdb

ABCC7 p.Met1137Ala

ClinVar:	c.3409A>G , p.Met1137Val ? , not provided c.3410T>G , p.Met1137Arg ? , not provided
CF databases:	c.3409A>G , p.Met1137Val (CFTR1) ? , This mutation (M1137V) in exon 18 of CFTR gene. The nucleotide at position 3541 was changed from A to G leading to a substitution of methionine codon for valine codon at position 1137. The mutation was foudn once in 384 chromsomes (289 CF chromosomes and 95 normal chromosomes) screened. Mutation on the other chromosome of the pancreatic sufficient patient is unknown. c.3410T>C , p.Met1137Thr (CFTR1) ? , The mutation was detected by DGGE analysis and characterized by direct sequencing. We have seen it only twice, in over 1300 control chromosomes from Italian population. c.3410T>G , p.Met1137Arg (CFTR1) ? , The M1137R mutation has been found once in 59 non-[delta]F508 chromosomes from the Portuguese population, associated with haplotype C. The patient carries the F1052V mutation on the other chromosome and presents a mild form of CF. M1137R was found neither in 28 normal chromosomes nor in 31 [delta]F508 CF chromosomes.
Predicted by SNAP2:	A: D (95%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (85%), K: D (95%), L: D (85%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: N (72%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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Publications
[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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No. Sentence Comment
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
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
95 Unitary Current Properties. Expression of wild-type, T1134A, M1137A, N1138A, and T1142A-CFTR in CHO cells led to the appearance of unitary PKAand ATP-dependent Cl-channel currents in excised membrane patches (Figure 3A). 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
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
119 Under steady-state conditions, each of those mutants which could be expressed in CHO cells appeared to have a lower mean channel open probability (PO) than wild-type, although this difference was only statistically significant for T1134A and M1137A (Figure 4B; P < 0.001, two-tailed t-test). 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
138 For wild-type, T1134A, and M1137A, data from these two different methods are in good agreement, however, for other TM12 mutants there is a significant discrepancy [(†) P < 0.01, (‡) P < 0.001, two-tailed t-test]. Login to comment
143 For wild-type CFTR, as well as for T1134A and M1137A, the inhibitory effects of 10 mM SCN- at -50 mV were the same at the macroscopic and single channel levels (Figure 5B). 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
164 However, as summarized in Table 1, TM12 mutations M1137A and N1138A did not alter the anion selectivity sequence, in stark contrast to the corresponding TM6 mutations F337A (20) and T338A (16). 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
184 Mean channel PO under steady-state conditions was reduced to 32% of wild-type levels in T1134A, and to 25% of wild-type in M1137A (Figures 3A and 4B). 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] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.

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212 In contrast, mutations in the analogous part of TM12 have been found to have little effect on conductance, which was reported as being unaltered in T1134A and M1137A [15] and slightly decreased in the less conservative T1134F [31]. 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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No. Sentence Comment
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