title: Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II.
abstract: The molecular mechanism underlying Ca(2+)/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 μm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 μm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln(855)-Glu(877)) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr(487) was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr(487) (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr(487) is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg(8)-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr(487) may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells.
authors.affiliation: Department of Physiology, Graduate School of Medical Sciences, Fukuoka University, Nanakuma 7-45-1, Johnan-ku, Fukuoka 814-0180, Japan.
authors.forename: Juan
authors.initials: J
authors.lastname: Shi
authors.forename: Naomi
authors.initials: N
authors.lastname: Geshi
authors.forename: Shinichi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kiyonaka
authors.forename: Jun
authors.initials: J
authors.lastname: Ichikawa
authors.forename: Yaopeng
authors.initials: Y
authors.lastname: Hu
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
authors.forename: Yushi
authors.initials: Y
authors.lastname: Ito
authors.forename: Ryuji
authors.initials: R
authors.lastname: Inoue
mesh_terms: D000200:Action Potentials; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D021381:Protein Transport; D050052:TRPC Cation Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
chemical_list: C503464:CaMKII inhibitor AIP; D010455:Peptides; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C492146:Trpc6 protein, mouse; D002217:Carbachol; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2