Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant

Abstract

The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) α-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.

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

“…1), even though this residue has been proposed to line a narrow region in the pore (25). Our previous work has failed to identify an important role for TM12 in formation of the narrow pore region (16,39).…”

“…However, functional evidence has indicated that TMs 1, 5, 6, 11, and 12 contribute to the lining of the pore and interact with Cl Ϫ ions (6,7,(13)(14)(15)(16). Extracellular loops between TMs 1 and 2 (ECL1) and TMs 11 and 12 (ECL6) have also been proposed to contribute to the outer mouth of the channel pore (15,17).…”

Metal Bridges Illuminate Transmembrane Domain Movements during Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

“…To investigate potential Cd 2ϩ bridges formed between porelining 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).…”

“…1). To investigate the potential for Cd 2ϩ bridge formation between pore-forming side chains coming from TMs 6 and 12, we introduced cysteines at each of four sites in TM6 (Ile-344, Val-345, Met-348, Ala-349) and seven sites in TM12 (Met-1140, Ser-1141, Thr-1142, Gln-1144, Trp-1145, Val-1147, Asn-1148) that we have previously found to be accessible to cysteine-reactive methanethiosulfonate reagents applied to the cytoplasmic side of the membrane (15,16). Combination of each single cysteine mutant from each TM then gave a total of 28 double-cysteine mutants containing one cysteine in TM6 and one cysteine in TM12 within the putative inner vestibule of the pore.…”

“…However, although experimental evidence for these different kinds of movement has been put forward, the relative mechanistic importance of these movements for the process of channel opening and closing is not known. Evidence for conformational changes in individual TMs within the inner vestibule of the pore during channel gating is mainly two-dimensional in nature, coming from state-dependent differences in the accessibility of cytoplasmically applied cysteine reactive methanethiosulfonate reagents to cysteine side chains introduced into TMs 1 (8,9,13,14), 6 (8, 9, 11, 15), 11 (10), and 12 (12,16). However, how the relative positions of these TM changes in three-dimensional space during channel gating have not previously been investigated.…”