Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPMT/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, Angus C. Nairn

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129 Citations (Scopus)

Abstract

TRPMT/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 TRPMT/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 Ser 1567, 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.

Original languageEnglish
Pages (from-to)20793-20803
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number21
DOIs
Publication statusPublished - May 27 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Matsushita, M., Kozak, J. A., Shimizu, Y., McLachlin, D. T., Yamaguchi, H., Wei, F. Y., Tomizawa, K., Matsui, H., Chait, B. T., Cahalan, M. D., & Nairn, A. C. (2005). Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPMT/ChaK1. Journal of Biological Chemistry, 280(21), 20793-20803. https://doi.org/10.1074/jbc.M413671200