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J. Biol. Chem. 276 (23): 20093-20100

© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Human CLC-3 Channels by Multifunctional Ca2+/Calmodulin-dependent Protein Kinase*

Ping HuangDagger , Jie LiuDagger , Anke Di, Nicole C. Robinson, Mark W. Musch§, Marcia A. Kaetzel, and Deborah J. Nelson||

From the Department of Neurobiology, Pharmacology and Physiology, § IBD Research Center and Department of Medicine, The University of Chicago, Chicago, Illinois 60637 and the  Department of Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267

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 (ICl,CaMKII), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of ICl,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.

* This work was supported by National Institutes of Health Grant GM36823 and a grant from the Cystic Fibrosis Foundation (Nelson96PO) (to D. J. N.), National Institutes of Health Grant DK46433 (to M. A. K.), National Institutes of Health Digestive Disease Core Grant DK-42086 and The Caroline Halfter Spahn Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to the fond memory of Wellesley Anne Johnson.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF172729.

Dagger Contributed equally to the results of this work.

|| To whom correspondence should be addressed: Dept. of Neurobiology, Pharmacology, and Physiology, the University of Chicago, 947 E. 58th St., MC 0926, Chicago, IL 60637. Tel.: 773-702-0126; Fax: 773-702-4066; E-mail: dnelson@drugs.bsd.uchicago.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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