Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Abstract

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl− channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84–T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

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

“…5, C and E), in E1371Q mutant channels, the K i for the double cysteine mutants was not significantly different from the single cysteine mutants (Fig. 5, B-E tional role lining the inner vestibule (3,13,14). However, in our hands, fewer side chains from TM1 line the inner vestibule (13), and when investigating potential Cd 2ϩ bridge formation involving this TM, we were limited to cysteines substituted at two positions (Lys-95 and Gln-98).…”

“…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.…”

“…Direct information concerning the three-dimensional structure of the pore has come from disulfide cross-linking experiments (7,13,17), as well as from experiments in which anionic channel blocker binding was altered by introduction of positively charged amino acid side chains (17,18). Previously, we have used the oxidizing agent copper(II)-o-phenanthroline to show that disulfide bonds can be formed between cysteine side chains introduced into different TMs, and we used this information to infer the relative proximity of residues in these different TMs lining the inner vestibule of the pore (13,17).…”

“…Previously, we have used the oxidizing agent copper(II)-o-phenanthroline to show that disulfide bonds can be formed between cysteine side chains introduced into different TMs, and we used this information to infer the relative proximity of residues in these different TMs lining the inner vestibule of the pore (13,17). However, because these disulfide bonds are essentially irreversible under non-reducing conditions, this approach has several disadvantages, such as the potential for disulfide bonds to form during rare or unnatural channel conformations when normally distant cysteine side chains transiently approach close together.…”

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

“…Based on functional data and homology models, CFTR has been predicted to contain five functional domains: two membranespanning domains (MSDs), each including six transmembrane (TM) helices; two nucleotide binding domains (NBD1 and NBD2); and a unique regulatory (R) domain, which carries multiple protein kinase A (PKA) consensus phosphorylation sites and is unique to CFTR in the ABC superfamily (1)(2)(3)(4)(5)(6). Functional studies from multiple groups have suggested that TM6 plays an essential role and TM12 contributes less to anion conduction and permeation properties in the CFTR channel pore (7)(8)(9)(10)(11).…”