Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

Key points center dot Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays pivotal roles in diverse Ca2+-mediated cellular functions including the physiology/pathophysiology of the cardiovascular system, through modulation of a variety of Ca2+-permeable channels such as a non-voltage-gated Ca2+ channel TRPC6. center dot In this study, we investigated the molecular mechanism underlying its positive regulation by CaMKII with chimera, deletion and site-directed mutagenesis approaches. center dot The results indicate that two spatially separated sites of TRPC6 channel, i.e. a distal part of the C-terminal inositol-1,4,5-trisphosphate receptor/CaM binding domain and Thr487 located on the putative first intracellular loop, are crucial for the CaMKII-mediated regulation of TRPC6 channels. center dot This mechanism may serve as an effective positive feedback regulation of Ca2+ influx through TRPC6 channels, in concert with intracellular and transmembrane Ca2+ mobilization upon phospholipase C-coupled receptor stimulation by neurohormonal factors, thereby fine-tuning the cardiovascular functions. center dot Disruption of these could lead to pathological states such as cardiac hypertrophy and arrhythmia, hypertension and atherosclerosis. Abstract The molecular mechanism underlying Ca2+/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 m) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 m). 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 (Gln855-Glu877) 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 Thr487 was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr487 (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 Thr487 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 Arg8-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 Thr487 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.