Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1

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 [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.