RRC ID 42333
Author Matsushita M, Kozak JA, Shimizu Y, McLachlin DT, Yamaguchi H, Wei FY, Tomizawa K, Matsui H, Chait BT, Cahalan MD, Nairn AC.
Title Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1.
Journal J Biol Chem
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.
Volume 280(21)
Pages 20793-803
Published 2005-5-27
DOI 10.1074/jbc.M413671200
PII S0021-9258(20)64084-4
PMID 15781465
MeSH Amino Acid Sequence Animals Binding Sites Calcium / metabolism Calcium / pharmacology Cations, Divalent Cell Line Crystallization Electric Conductivity Escherichia coli / genetics Gene Expression Ion Channels / chemistry Ion Channels / genetics Ion Channels / physiology* Magnesium / pharmacology Mass Spectrometry Membrane Proteins / chemistry Membrane Proteins / genetics Membrane Proteins / physiology* Mice Molecular Sequence Data Mutagenesis, Site-Directed Patch-Clamp Techniques Phosphorylation Protein Kinases / chemistry Protein Kinases / genetics Protein Kinases / metabolism* Protein Kinases / physiology* Recombinant Fusion Proteins Spodoptera Structure-Activity Relationship TRPM Cation Channels Zinc / pharmacology
IF 4.238
Times Cited 125
Human and Animal Cells