Role of endogenous TRPC6 channels in Ca²⁺ signal generation in A7r5 smooth muscle cells

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca²⁺ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn- glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca²⁺ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca²⁺ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca²⁺ entry was suppressed by more than 50%. In contrast, OAG-induced Ca²⁺ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca²⁺ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2). Importantly, OAG-induced Ca²⁺ entry was blocked by the potent L-type Ca²⁺ channel inhibitor nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca²⁺ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.