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.
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Title |
Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPM7/ChaK1.
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Journal |
J Biol Chem
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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
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Published |
2005-5-27
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DOI |
10.1074/jbc.M413671200
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PII |
S0021-9258(20)64084-4
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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
|
WOS Category
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
Resource |
Human and Animal Cells |
|