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 [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.
Matsushita
Masayuki
M
First Department of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Kozak
J Ashot
JA
Shimizu
Yoshio
Y
McLachlin
Derek T
DT
Yamaguchi
Hiroto
H
Wei
Fan-Yan
FY
Tomizawa
Kazuhito
K
Matsui
Hideki
H
Chait
Brian T
BT
Cahalan
Michael D
MD
Nairn
Angus C
AC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent; D002460:Cell Line; D003460:Crystallization; D004553:Electric Conductivity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D018411:Spodoptera; D013329:Structure-Activity Relationship; D050053:TRPM Cation Channels; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D007473:Ion Channels; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels; D011494:Protein Kinases; C421903:Trpm7 protein, mouse; D008274:Magnesium; D015032:Zinc; D002118:Calcium