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 CIC 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,I-CaMKII), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I-Cl,I-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-de pendent 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.