A Monomeric Variant of the CLC-ec1 Transporter

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CLC-ec1, a prototypical Cl-/H+ exchanger of the CLC family, is a homodimer in which each subunit is thought to act as a fully functional transporter. Why then is this protein a dimer and what are the factors driving dimer formation? In this study, we address these questions by destabilizing the CLC-ec1 subunit-subunit interface. We substituted tryptophan residues to create steric mismatches at the interface that would also favor lipid interactions. A single mutation, I422W, was sufficient to destabilize the CLC-ec1 complex resulting in a shift from dimer to monomer position on gel-filtration chromatography. Glutaraldehyde cross-linking of I422W in both detergent and phosphatidyl-choline/phosphatidyl-glycerol liposomes showed that the mutant remained a monomer compared to the wild-type protein; however dimer formation was observable at high concentrations. The I422W mutant was functional as measured by 36Cl- uptake, passive Cl- efflux and Cl- driven H+ pumping in E. coli phospholipids. A Poisson-counting method demonstrated that I422W re-dimerizes in these phosphatidyl-ethanolamine-rich liposomes, even at very low protein/lipid ratio. Altogether, these findings suggest that we have purified an isolated monomeric variant of CLC-ec1 that demonstrates shifted dimerization energetics depending on the lipid environment. We continue to search for mutations that will more severely destabilize the dimer, in attempts to obtain a transport-competent, monomeric CLC exchanger.