ABCMdb

ABCC7 p.Asn1138Cys

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] 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. Login to comment
59 a Example time courses of macroscopic currents (measured at -80 mV) carried by N1138C and T1142C as indicated in inside-out membrane patches. Login to comment
62 b Example leak subtracted I-V relationships for N1138C, T1142C, V1147C, and N1148C, recorded from inside out membrane patches following maximal channel activation with PKA, ATP, and PPi. Login to comment
64 As described in the text, whereas MTSES application always led to a decrease in macroscopic current amplitude in reactive mutants, the effects of MTSET were to decrease (e.g., N1138C, V1147C), increase (e.g., T1142C) or not significantly alter (e.g., N1148C) macroscopic current amplitude. Login to comment
80 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). Login to comment
82 In the central region of TM12 (N1138C, M1140C, and S1141C), macroscopic current amplitude was decreased by both MTSES and MTSET (Figs. 1 and 2). Login to comment
92 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). Login to comment
93 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. Login to comment
94 The overall pattern of calculated modification rate constants shown in Fig. 3b suggests that modification is faster for cysteines introduced closer to the cytoplasmic end of TM12 and slower at N1138C located further along the axis of TM12. Login to comment
96 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. Login to comment
101 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. Login to comment
103 These results suggest that none of N1138C, M1140C, or S1141C can be modified covalently by extracellular MTSET. Login to comment
106 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. Login to comment
119 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. Login to comment
120 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. Login to comment
125 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. Login to comment
140 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]. Login to comment
143 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. Login to comment
148 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). Login to comment

[hide] Structural basis for the channel function of a deg... J Gen Physiol. 2011 Nov;138(5):495-507. Bai Y, Li M, Hwang TC
Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).
J Gen Physiol. 2011 Nov;138(5):495-507., [PMID:22042986]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.

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No. Sentence Comment
209 However, although they detected reactivity in N1138C and S1149C, we found that these sites appear nonreactive. Login to comment