Abstract
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 SO(4)(2-) 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 Glu(in) 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 Glu(in) site in CLC-ck2 does not increase H(+) flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu(ex)) in CLC-ck2 prevents H(+) flux. Hence, Glu(ex), but not Glu(in), is critical for H(+) permeation in CLC proteins.