CLC Cl /H+ transporters constrained by covalent cross-linking.
CLC Cl(-)/H(+) exchangers are homodimers with Cl(-)-binding and H(+)-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine-lysine cross-bridge was formed with I(2) as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl(-) turnover rate, Cl(-)/H(+) exchange stoichiometry, and H(+) pumping driven by a Cl(-) gradient. These results imply that large quaternary rearrangements, such as those known to occur for "common gating" in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with "fast gating" of the CLC channels.
Nguitragool
Wang
W
Department of Biochemistry, Howard Hughes Medical Institute and Brandeis University, Waltham, MA 02454, USA.
Miller
Christopher
C
D000818:Animals; D001692:Biological Transport; D018118:Chloride Channels / Q000737:chemistry / Q000502:physiology*; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008968:Molecular Conformation; D009154:Mutation; D016877:Oxidants / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D017433:Protein Structure, Secondary
C086321:CLC-1 channel; D018118:Chloride Channels; D003432:Cross-Linking Reagents; D004220:Disulfides; D007477:Ions; D008051:Lipid Bilayers; D016877:Oxidants; D003553:Cystine; D008239:Lysine; D003545:Cysteine; D010100:Oxygen