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,Angus C Nairn

doi:10.1074/jbc.m413671200

Abstract

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 Ser(1511) 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 [Mg(2+)]. 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 Ca(2+) influx. Inhibition by internal Mg(2+) was also unaffected in the autophosphorylation site or "kinase-dead" mutants. Moreover, kinase activity was enhanced by Mg(2+), was decreased by Zn(2+), and was unaffected by Ca(2+). 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 Mg(2+) is affected by kinase activity or autophosphorylation but that the kinase domain may play a structural role in channel assembly or subcellular localization.

Highlights

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
Several additional catalytic domains related to elongation factor-2 kinase (EF2K) and MHCK have been identified through data base searches in mammals as well as in nematode worm, the function of these potential protein kinases is not known [1,2,3]
Characterization of the Kinase Activity and Autophosphorylation of TRPM7—To examine the kinase activity of TRPM7, several GST fusion proteins containing the kinase domain and additional N-terminal extensions were expressed in E. coli and purified (Fig. 1 and data not shown)

Summary

Introduction

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. Several additional catalytic domains related to EF2K and MHCK have been identified through data base searches in mammals as well as in nematode worm, the function of these potential protein kinases is not known [1,2,3] Using this approach, we and others identified an 1863-amino acid polypeptide (termed ChaK for channel kinase) that contained a TRP-related channel domain fused to the atypical kinase domain at its C terminus [3, 4]. In contrast to the classical kinase superfamily, the C-terminal lobe of the TRPM7 kinase domain contains a zinc finger homology domain, and zinc appears to play a structural role in TRPM7 and related EF2K/MHCK family members [4]. Similar currents have been described in cardiac fibroblasts [17], smooth muscle cells [18], and brain microglia [19]

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