Referenced in: none

Automatically associated channels: ClC4

Title: Inaugural Article: CLC Cl /H+ transporters constrained by covalent cross-linking.

Authors: Wang Nguitragool, Christopher Miller

Journal, date & volume: Proc. Natl. Acad. Sci. U.S.A., 2007 Dec 26 , 104, 20659-65

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/18093952

Abstract
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.