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Splice variation of the cytoplasmic C‐terminus of a C. elegans ClC channel alters functional properties and glutamate gate accessibility to extracellular ions
Author(s) -
He liping,
Denton Jerod,
Nehrke Keith,
Strange kevin
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a324-b
Subject(s) - cysteine , gating , splice , amino acid , extracellular , glutamic acid , cytoplasm , biology , glutamate receptor , chemistry , c terminus , microbiology and biotechnology , biophysics , biochemistry , gene , enzyme , receptor
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.