title: 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.
authors.affiliation: Department of Physiology & Biophysics, Dalhousie University, 1459 Oxford Street, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Feng
authors.initials: F
authors.lastname: Qian
authors.forename: Yassine
authors.initials: Y
authors.lastname: El Hiani
authors.forename: Paul
authors.initials: P
authors.lastname: Linsdell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008698:Mesylates / Q000494:pharmacology; D018408:Patch-Clamp Techniques
chemical_list: D008698:Mesylates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C074502:methanethiosulfonate; D003545:Cysteine