Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

doi:10.1113/jphysiol.2003.058032

Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current

^¹Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, IL 60637, USA^²Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH 45267, USA^³Department of Pediatrics, University of Iowa, Iowa City, IA 52242, Chicago, IL 60637, USA

Corresponding author D. J. Nelson: Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, 947 East 58^th Street, AB-500 MC-0926, Chicago, IL 60637, USA. Email: dnelson{at}drugs.bsd.uchicago.edu

Abstract

CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium–calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl⁻ conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl⁻ conductance was not observed following exposure of the cells to 1 μm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl⁻ conductance; however, CLC-3^−/− cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl⁻ conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane.