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Identification of an N‐terminal amino acid of the CLC‐3 chloride channel critical in phosphorylation‐dependent activation of a CaMKII‐activated chloride current
Author(s) -
Robinson N. C.,
Huang P.,
Kaetzel M. A.,
Lamb Fred S.,
Nelson D. J.
Publication year - 2004
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2003.058032
Subject(s) - chloride channel , microbiology and biotechnology , chemistry , intracellular , patch clamp , phosphorylation , gating , hek 293 cells , biochemistry , biophysics , biology , receptor
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 μ m 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.