Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells

We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 muM)-activated TRPC6 currents (I-TRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca-o(2+)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca-o(2+) solely inhibited TRPC7 currents (I-TRPC7). Vigorous buffering of intracellular Ca2+ (Ca-i(2+)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca-o(2+), disclosing fast potentiation (EC50: similar to0.4 mm) and inhibition (IC50: 4 mm) of I-TRPC6 and fast inhibition (IC50: similar to0.4 mm) of I-TRPC7. This inhibition of I-TRPC6 and I-TRPC7 seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I-TRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I-TRPC6 or its acceleration by Ca2+ probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 mum), coexpression of Ca2+-insesentive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I-TRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.