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J. Biol. Chem., Vol. 280, Issue 21, 20793-20803, May 27, 2005

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Channel Function Is Dissociated from the Intrinsic Kinase Activity and Autophosphorylation of TRPM7/ChaK1^*

Masayuki Matsushita, J. Ashot Kozak¶, Yoshio Shimizu||, Derek T. McLachlin**, Hiroto Yamaguchi, Fan-Yan Wei, Kazuhito Tomizawa, Hideki Matsui, Brian T. Chait**, Michael D. Cahalan, and Angus C. Nairn||¶¶||||

From the First Department of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan, the Department of Structure Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma Nara 630-0101, Japan, ^||Laboratory of Molecular and Cellular Neuroscience and ^**Laboratory of Mass Spectrometry, The Rockefeller University, New York, New York 10021, the Department of Physiology and Biophysics, University of California, Irvine, California 92697-4561, and the ^¶¶Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06508

TRPM7/ChaK1 is a unique channel/kinase that contains a TRPM channel domain with 6 transmembrane segments fused to a novel serine-threonine kinase domain at its C terminus. The goal of this study was to investigate a possible role of kinase activity and autophosphorylation in regulation of channel activity of TRPM7/ChaK1. Residues essential for kinase activity were identified by site-directed mutagenesis. Two major sites of autophosphorylation were identified in vitro by mass spectrometry at Ser¹⁵¹¹ and Ser¹⁵⁶⁷, and these sites were found to be phosphorylated in intact cells. TRPM7/ChaK1 is a cation-selective channel that exhibits strong outward rectification and inhibition by millimolar levels of internal [Mg²⁺]. Mutation of the two autophosphorylation sites or of a key catalytic site that abolished kinase activity did not alter channel activity measured by whole-cell recording or Ca²⁺ influx. Inhibition by internal Mg²⁺ was also unaffected in the autophosphorylation site or "kinase-dead" mutants. Moreover, kinase activity was enhanced by Mg²⁺, was decreased by Zn²⁺, and was unaffected by Ca²⁺. In contrast, channel activity was inhibited by all three of these divalent cations. However, deletion of much of C-terminal kinase domain resulted in expression of an apparently inactive channel. We conclude that neither current activity nor regulation by internal Mg²⁺ is affected by kinase activity or autophosphorylation but that the kinase domain may play a structural role in channel assembly or subcellular localization.

Received for publication, December 6, 2004 , and in revised form, February 22, 2005.

^* This work was supported by United States Public Health Service Grants GM50402 (to A. C. N.), NS14609 (to M. D. C.), and RR00862 (to B. T. C.) and by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of an American Heart Association Postdoctoral Fellowship.

Recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research.

^|||| To whom correspondence should be addressed: Dept. of Psychiatry, Yale University School of Medicine, New Haven, CT 06508. Tel.: 203-974-7725; Fax: 203-974-7724; E-mail: angus.nairn{at}yale.edu.

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