Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current.
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 microm 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.
Robinson
N C
NC
Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, 947 East 58(th) Street, AB-500 MC-0926, Chicago, IL 60637, USA.
Huang
P
P
Kaetzel
M A
MA
Lamb
Fred S
FS
Nelson
D J
DJ
D000818:Animals; D001011:Aorta / Q000166:cytology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D008817:Mice, Mutant Strains; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D014162:Transfection
D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases