CLC Cl ⁻ /H ⁺ transporters constrained by covalent cross-linking

Abstract

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on May 1, 2007.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.disulfide ͉ oxidation ͉ sulfenamide ͉ antiporter ͉ exchanger

Ion permeation through a Cl − -selective channel designed from a CLC Cl − /H + exchanger

“…Transport rate was calculated from the initial rate of Cl Ϫ release (ions per second), determined in each experiment by calibrating with KCl addition. All fluxes are reported as Cl Ϫ ions per second per CLC-ec1 subunit, which yields values 50% of those reported previously because recent results demonstrate that the homodimeric complex contains two transporters, one in each subunit (19).…”

“…These ingredients were stored in aliquots at Ϫ80°C, and after thawing on the day of the flux assay they were passed through a 400-nm extrusion apparatus to form unilamellar vesicles. In some cases, samples were prepared for electrical recording in planar lipid bilayers, as described (19,22); all mutants with substitutions at Glu ex that were tested in planar bilayers were ideally Cl Ϫ -selective, as expected (38).…”

“…Unitary transport rates were determined by a Poisson dilution method (19,22) with a concentration of protein so low that most of the transporting liposomes contained only a single CLC-ec1 homodimer. Most experiments were done on liposomes made from separate reconstitutions from the same protein preparation, but in some case several protein preparations were also used.…”

“…The protein is a homodimer (Fig. 1) in which each subunit acts as a full-fledged transporter (19). Each subunit contains multiple ion-binding sites located on structurally delineated transmembrane ''pathways'' along which Cl Ϫ and H ϩ move in opposite directions.…”

Charge Transport in the ClC-type Chloride-Proton Anti-porter from Escherichia coli

“…Bookkeeping of EClC Microstates-The transport mechanism in both monomers of the homo-dimeric EClC is likely not directly coupled (21). Therefore, we used a simplified description of EClC function assuming that both monomers adopt the same state.…”