Identification of a regulatory phosphorylation site in a cell cycle and swelling regulated ClC Cl‐ channel

Abstract

clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans voltage‐gated ClC Cl‐ channel. CLH‐3b is expressed in the worm oocyte and is activated during meiotic cell cycle progression or in response to cell swelling. Channel activation is brought about by serine/threonine dephosphorylation mediated by the type 1 phosphatases GLC‐7α and GLC‐7β. GCK‐3 is a Ste20 kinase and homolog of mammalian PASK/SPAK, which regulates cation‐coupled Cl‐ cotransporters. GCK‐3 binds to a 101 amino acid C‐terminal splice insert unique to CLH‐3b. When co‐expressed with CLH‐3b, GCK‐3 dramatically inhibits channel activity and alters gating kinetics and voltage sensitivity. Knockdown of GCK‐3 in worm oocytes constitutively activates CLH‐3b. Using mass spectrometry, we identified a serine residue (S747) located within the 101 amino acid splice insert that is phosphorylated in the presence of GCK‐3. S747 and surrounding amino acids conform to the Ste20 kinase phosphorylation motif. Mutation of S747 to alanine prevents GCK‐3 mediated channel inhibition and alterations of channel voltage‐dependent properties. We conclude that phosphorylation of S747 is critical for regulation of CLH‐3b activity. S747 may be phosphorylated directly by GCK‐3 or other downstream kinases.Supported by NIH R01 grant DK51610