ABCC7 p.Met1140Cys
| ClinVar: |
c.3419T>A
,
p.Met1140Lys
?
, not provided
|
| CF databases: |
c.3419T>A
,
p.Met1140Lys
(CFTR1)
?
, Found in a patient with bronchiectasis.
|
| Predicted by SNAP2: | A: D (85%), C: D (91%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (95%), I: D (91%), K: D (95%), L: D (85%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (91%), V: D (85%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[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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No. Sentence Comment
5 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.
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ABCC7 p.Met1140Cys 21796338:5:130
status: NEW80 Application of MTSES (200 μM) or MTSET (2 mM) to the intracellular solution after channel activation with PKA, ATP, and PPi significantly altered macroscopic current amplitude in nine out of 19 cysteine-substituted mutants tested (N1138C, M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C, and S1149C; Figs. 1 and 2).
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ABCC7 p.Met1140Cys 21796338:80:245
status: NEW82 In the central region of TM12 (N1138C, M1140C, and S1141C), macroscopic current amplitude was decreased by both MTSES and MTSET (Figs. 1 and 2).
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ABCC7 p.Met1140Cys 21796338:82:39
status: NEW92 For MTS reagent-sensitive TM12 mutants located relatively deeply into the pore from its cytoplasmic end (N1138C, M1140C, S1141C, and T1142C), the rate of modification was estimated from the time course of macroscopic current amplitude change following application of MTSES (20-200 μM).
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ABCC7 p.Met1140Cys 21796338:92:113
status: NEW93 As shown in Fig. 3a, modification was rapid in M1140C, S1141C, and T1142C, even using a low concentration of MTSES (20 μM), and noticeably slower in N1138C, even with 200 μM MTSES.
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ABCC7 p.Met1140Cys 21796338:93:47
status: NEW96 To gain some information on the orientation of TM12 in the CFTR pore, we therefore examined whether the outermost cysteines introduced into this TM that were sensitive to internal MTS reagents (N1138C, M1140C, and S1141C) could also be modified by extracellular MTSET.
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ABCC7 p.Met1140Cys 21796338:96:202
status: NEW101 As shown in Fig. 4a, patches excised from MTSET-pretreated cells expressing N1138C, M1140C, or S1141C all gave macroscopic currents that were decreased in amplitude following addition of 2 mM MTSET to the intracellular solution.
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ABCC7 p.Met1140Cys 21796338:101:84
status: NEW103 These results suggest that none of N1138C, M1140C, or S1141C can be modified covalently by extracellular MTSET.
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ABCC7 p.Met1140Cys 21796338:103:43
status: NEW106 We used a similar approach to determine whether N1138C, M1140C, S1141C, and T1142C, located relatively deeply into the pore from its cytoplasmic end and all strongly sensitive to inhibition by intracellular MTSES (Figs. 2 and 3), could be modified by MTSES pretreatment.
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ABCC7 p.Met1140Cys 21796338:106:56
status: NEW119 In contrast, currents carried by both N1138C and M1140C were strongly inhibited by application of the test dose of MTSES, suggesting that they had not been covalently modified by MTSES pretreatment.
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ABCC7 p.Met1140Cys 21796338:119:49
status: NEW120 These results, which are summarized quantitatively in Fig. 5d, suggest that while S1141C and T1142C can be modified by MTSES prior to channel activation, N1138C and M1140C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP.
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ABCC7 p.Met1140Cys 21796338:120:165
status: NEW125 Extracellular MTSET did not appear able to modify the outermost of these internal MTS-sensitive cysteines (N1138C, M1140C, and S1141C; Fig. 4), consistent with MTS reagents not being permeant.
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ABCC7 p.Met1140Cys 21796338:125:115
status: NEW140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9].
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ABCC7 p.Met1140Cys 21796338:140:262
status: NEW143 a Example timecourses of macroscopic current amplitudes (measured at -50 mV) carried by N1138C, M1140C, S1141C, and T1142C as indicated in inside-out membrane patches.
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ABCC7 p.Met1140Cys 21796338:143:96
status: NEW148 Using a similar approach, we find that in TM12, S1141C and T1142C can be readily modified by cytoplasmic MTSES prior to channel activation (Fig. 5), whereas N1138C and M1140C are modified rapidly after channel activation (Fig. 3) but very slowly, if at all, prior to channel activation (Fig. 5).
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ABCC7 p.Met1140Cys 21796338:148:168
status: NEW[hide] Metal bridges illuminate transmembrane domain move... J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20. El Hiani Y, Linsdell P
Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20., [PMID:25143385]
Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane alpha-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.
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None has been submitted yet.
No. Sentence Comment
51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12).
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ABCC7 p.Met1140Cys 25143385:51:381
status: NEW128 Also note that the estimated Ki values for some double mutants were c56;300 òe;M (V345C/M1140C, 316 afe; 38 òe;M, n afd; 3; A349C/M1140C, 345 afe; 58 òe;M, n afd; 3; A349C/T1142C, 231 afe; 68 òe;M, n afd; 3).
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ABCC7 p.Met1140Cys 25143385:128:95
status: NEWX
ABCC7 p.Met1140Cys 25143385:128:147
status: NEW