ABCMdb

ABCC7 p.Asn1138Ala

Predicted by SNAP2:	A: D (63%), C: D (75%), D: D (80%), E: D (85%), F: D (85%), G: D (75%), H: D (85%), I: D (75%), K: D (80%), L: D (71%), M: D (75%), P: D (80%), Q: D (63%), R: D (85%), S: N (61%), T: N (66%), V: D (75%), W: D (91%), Y: D (80%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, P: D, Q: N, R: N, S: 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
377 N1138A and T1142A significantly blocked the channel by indirectly reducing interaction between ClÀ ions and glibenclamide within the pore [179]. 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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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
102 For each of the five TM12 mutants, the form of the i-V relationship was very similar to that for wild-type (e.g., N1138A; Figure 3B), and the mean slope conductance was not significantly different to that of wild-type (7.8 ( 0.3 pS; n ) 28; Figure 4A). 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
115 (B) Mean single channel current-voltage relationship for wild-type (O) and N1138A (b). 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
176 This effect did not appear to be a nonspecific result of mutagenesis within TM12, as SCN- block was unaltered in both N1138A and T1142A. Login to comment
185 A similar trend was observed in N1138A and T1142A (Figure 4B), although in these cases the reduction in mean PO was not statistically significant (0.05 < P < 0.15 in both cases, two-tailed t-test). 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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5 Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. Login to 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
69 Mean fraction of control current remaining following addition of 60 µM glibenclamide (I/I0) is shown as a function of voltage for wild-type (q), T338A (s), N1138A (s), F337A (ss) and T1142A (xx). Login to comment
70 Mean of data from 5-10 patches, fitted by Eq. according to the mean parameters shown in Fig. 3 rent remaining following addition of glibenclamide (I/I0) was significantly reduced at all voltages in N1138A and T1142A (P<0.05), and significantly increased in F337A and T338A at negative membrane potentials (P<0.05). Login to comment
75 Consistent with the results shown in Fig. 2, Kd(0) was significantly increased in F337A and T338A, and significantly decreased in N1138A and T1142A (Fig. 3A). Login to comment
84 In contrast, reducing extracellular Cl-concentration from 154 mM to 10 mM had no significant effect on apparent glibenclamide affinity in the TM12 mutants N1138A (Fig. 5A) or T1142A (Figs. 4, 5A). Login to comment
85 As a result, the effect of these two mutations on Kd(0) was dependent on the extracellular Cl-concentration: in N1138A, Kd(0) was reduced to 35% of the wild-type value with 154 mM Cl- but only to 61% of wild-type with 10 mM Cl-, while in T1142A, Kd(0) was 40% of wild-type with 154 mM Cl- and 50% with 10 mM Cl-. Login to comment
100 A similar lack of extracellular Cl-dependence was observed in N1138A (data not shown) Discussion Glibenclamide causes a relatively high affinity block of CFTR [32, 34] and other Cl-channels [14, 29, 31, 42]. Login to comment
115 In contrast to the effects of these mutations in TM6, two mutations in TM12 (N1138A and T1142A) caused a significant increase in the apparent affinity of glibenclamide block (Figs. 2, 3). Login to comment
120 Two kinds of interactions may underlie the ability of extracellular Cl- ions to weaken glibenclamide block of wild-type, but not N1138A or T1142A CFTR. Login to comment
122 In this case, N1138A and T1142A might alter the structure of a binding site for both Cl- and glibenclamide, reducing its affinity for Cl- ions and thereby indirectly increasing glibenclamide occupancy of the pore. Login to comment
132 However, it is interesting to note that both suggest that: (1) the mutations N1138A and T1142A can increase the apparent affinity of glibenclamide block without directly affecting the interaction between glibenclamide and the pore walls, and (2) these two mutations both interfere with Cl-binding within the pore. Login to comment
134 Furthermore, the suggestion that TM12 residues N1138 and T1142 contribute to Cl-binding within the pore conflicts with our previous finding that the mutations N1138A and T1142A have no effect on unitary Cl-conductance [10]. Login to comment

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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207 Finally, two mutations of TM12 residues that also project towards the pore (N1138A and T1142A, Fig. 3B) were reported to significantly strengthen the glibenclamide block and to abolish its dependence on the extracellular Cl-concentration [69], suggesting that these mutations may alter interactions between glibenclamide and Cl- ions within the pore. 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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150 Surprisingly, nine mutations of TM12, including N1138A, M1140A, T1142A, V1147A, N1148A, S1149A, S1150A, I1151A, and D1152A, exhibited significantly altered block by Glyb; the pattern was not consistent with either α-helix or β-strand secondary structure along the full length of the region studied. Login to 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