Surprises from an Unusual CLC Homolog

The chloride channel (CLC) family is distinctive in that some members are Cl− ion channels and others are Cl−/H+ antiporters. The molecular mechanism that couples H+ and Cl− transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl− and H+ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl−-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl− transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO42− strongly competes with Cl− uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl−/H+ antiporter, even though it contains an isoleucine at the Gluin position that was previously thought to be a critical part of the H+ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Gluin site in CLC-ck2 does not increase H+ flux. Like other CLC antiporters, mutation of the external glutamate gate (Gluex) in CLC-ck2 prevents H+ flux. Hence, Gluex, but not Gluin, is critical for H+ permeation in CLC proteins.