title: Inter-subunit communication and fast gate integrity are important for common gating in hClC-1.
abstract: Proteins of the CLC family are comprised of two subunits, each with its own fast-gated protopore, both of these being regulated simultaneously by a slower common gate. Based on the X-ray crystal structure of a bacterial CLC, the carboxyl side chain of glutamate residue E232 has been proposed as the fast gate of hClC-1, swinging into each pore to close it and competing with chloride. We now show, using hClC-1 mutants expressed in whole-cell patch-clamped HEK293 cells, that elimination of this side chain in the E232Q mutation prevents fast gate closure at all voltages but common gating is also eliminated suggesting that E232 could be the final effector of both fast and common gating. We hypothesise that the conformational information essential for common gating flows between the two E232 protopore residues across the intra-membrane interface, rather than via any cytoplasmic carboxyl-tail interface, to drive common gating. Informed by in silico modelling, we have produced five site-directed mutants that increase the volumes of residues which might be involved in allosteric transfer (A272V, A272L, S289L, V292L and T293L) and assessed them for effects on gating. These mutations could be expected to increase molecular forces between, or torques around, the intimate L287-L287 and I290-I290 contacts that form the pseudo-asymmetric axis of the hClC-1 dimer. Common gating is practically eliminated in V292L and open probability is shifted to more depolarised potentials in A272V, S289L and T293L mainly by altering the voltage dependence of common gating.
authors.affiliation: Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, North Terrace, Adelaide, SA 5000, Australia.
authors.forename: Jennie M
authors.initials: JM
authors.lastname: Cederholm
authors.forename: Grigori Y
authors.initials: GY
authors.lastname: Rychkov
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Bagley
authors.forename: Allan H
authors.initials: AH
authors.lastname: Bretag
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D050505:Mutant Proteins / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*
chemical_list: C086321:CLC-1 channel; D018118:Chloride Channels; D050505:Mutant Proteins; D021122:Protein Subunits