Splice variation of the cytoplasmic C‐terminus of a C. elegans ClC channel alters functional properties and glutamate gate accessibility to extracellular ions

Abstract

CLH-3a and CLH-3b are ClC splice variants that exhibit striking differences in voltage-dependent gating and bath Cl− and H+ sensitivity. The proteins have identical predicted intramembrane domains, but differ significantly in their cytoplasmic N- and C-termini. We postulated that splice variation of the N- and/or C-termini alters outer pore structure, which in turn gives rise to the unique functional properties of the two channels. Deletion of the unique N-terminus of CLH-3a or addition of this domain to CLH-3b had no effect on channel properties. In contrast, deletion of the unique 169 amino acid C-terminus of CLH-3b, or deletion of the last 11 amino acids of CBS domain 1 gave rise to channel properties that fully recapitulated those of CLH-3a. Mutation of the extracellular glutamate gate in both channels to alanine or cysteine abolished voltage and bath Cl− and H+ sensitivity. Exposure to MTSET inhibited both cysteine mutants ~70%. Mean MTSET inhibition time constants were 24 sec and 144 sec for CLH-3a(E238C) and CLH-3b(E167C), respectively. Deletion mutagenesis of the CLH-3b C-terminus or CBS domain 1 increased the accessibility of the engineered cysteine residue to a level that was indistinguishable from CLH-3a(E238C). We conclude that splice variation of the CLH-3 cytoplasmic C-terminus alters voltage-dependent gating and bath ion sensitivity by altering the structural properties of the outer pore vestibule. CBS domain 1 also modulates outer pore structure, possibly through a functional interaction with the distal C-terminus. Supported by NIH grant DK51160.