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GCK‐3 induced phosphorylation alters ClC anion channel outer pore structure
Author(s) -
Bhate Manasi,
He liping,
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-c
Subject(s) - extracellular , intracellular , gating , chemistry , phosphorylation , cysteine , biophysics , glutamate receptor , microbiology and biotechnology , kinase , biochemistry , mutant , biology , receptor , enzyme , gene
ClC anion channels are homodimeric proteins that play key physiological roles in organisms from bacteria to humans. ClC monomers form independently gated protopores and are comprised of intracellular N‐ and C‐termini and 18 α‐helical domains termed helices A‐R. Helices D, F, N and R contribute amino acids that mediate Cl − binding within the outer pore vestibule as well as an extracellular facing glutamate residue or “gate” that opens and closes the protopore. In eukaryotes, helix R is connected to a large cytoplasmic C‐terminus that could provide a route for regulating ClC protopore gating by cellular signaling events. CLH‐3b is a C. elegans ClC channel that is inhibited by GCK‐3, a Ste20 kinase that binds to the channel C‐terminus. We postulated that GCK‐3 inhibits CLH‐3b by altering the structural relationships of the extracellular glutamate gate and associated selectivity filter. To test this idea, we mutated the glutamate gate to cysteine (E167C) and assessed the effect of extracellular exposure to the sulfhydryl reagent MTSET. As expected, E167C channels are constitutively active. MTSET inhibited E167C activity with a time constant of 129 ± 12 sec. Co‐expression of E167C with GCK‐3 dramatically reduced the inhibition time constant to 25 ± 3 sec. In contrast, a kinase‐dead GCK‐3 mutant had no effect on MTSET inhibition. These data suggest that GCK‐3 mediated phosphorylation regulates CLH‐3b activity by modifying the structure of the outer pore vestibule. Our studies provide the first direct demonstration that intracellular signaling events alter the structure/function relationships of ClC channels. Supported by NIH grant DK51610 and an APS Undergraduate Summer Research Fellowship.