Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase.
The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.
Department of Neurobiology, Pharmacology and Physiology, IBD Research Center and Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
P
P
Huang
J
J
Liu
A
A
Di
N C
NC
Robinson
M W
MW
Musch
M A
MA
Kaetzel
D J
DJ
Nelson
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018118:Chloride Channels; C115376:ClC-3 channel; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases