Differential Regulation By Calcium Of Two Closely Related TRP Homologues TRPC6 And TRPC7

Elucidation of activation and regulatory mechanisms for non-voltage-gated Ca2+ entry is an essential step toward understanding diverse Ca2+ signaling in both excitable and non-excitable cells. Here we describe that by using heterologous expression and patch clamp techniques, Ca2+/calmodulin (CaM) play contrasting roles in regulating TRPC6 and TRPC7, two closely related members of TRP (transient receptor potential) channel family that has recently received much attention as plausible molecular candidates for receptor-activated Ca2+ entry channels. In the nystatin-perforated mode, macroscopic TRPC6 currents evoked by carbachol (CCh; 100M) exhibited bi-phasic responses to extracellularly applied Ca2+(Ca2+0; 1mM); fast potentiation followed by sustained inhibition. In contrast, CCh-evoked TRPC7 currents underwent solely a fast dose-dependent inhibition by Ca2+. Introduction of 10 mM BAPTA into the cell abolished the inhibition of TRPC6 by Ca2+. rendering the potentiative effect sustained, while only moderately relieved the inhibition of TRPC7. In inside-out patch recording, single TRPC6 channel activities characterized by brief openings (t=2.7 ms) were observed in the presence of CCh (100M) in the pipette. These channel activities were greatly enhanced and changed to a long-lasting pattern (t=30 ms) when the cytosolic concentration of Ca2+ ([Ca2+]i) was raised to the micromolar range, while only a moderate decrease was observed at nanomolar concentrations of [Ca+]i. In sharp contrast with TRPC6, single CCh-activated TRPC7 channels were dose-dependently inhibited by increasing [Ca2+]i in the nanomolar range, and no discernible potentiation occurred in the micromolar range of [Ca2+]i.The effects of intracellular Ca2+ on TRPC6 and TRPC7 were abolished by pretreatment with a calmodulin antagonist calmidazolium (CMZ) or co-expression of a Ca2+-insensitive form of calmodulin with four aspartate-to-alanine substitutions. Experiments using various chimeras of TRPC6 and TRPC7 revealed that the fast inhibition of TRPC7 and potentiation of TRPC6 by Ca2+ depends specifically on their transmembrane regions, whereas the reciprocal regulation of TRPC6 and TRPC7 via Ca2+/CaM is critically determined by the C-terminal difference. In addition, in about a 50% of patch membranes tested, single TRPC7 channel activities were enhanced by adding IP3 (10M) at the cytosolic side, which was not evident for TRPC6. From these results, we conclude that TRPC7 channel undergoes Ca2+-mediated inhibition from both external and internal sides of the membrane; and the former probably results from ion permeation blockade by Ca2+ near the outer mouth of ion conductive pore, while the latter likely involves Ca2+-dependent tonic inhibition by CaM on the intracellular C-terminus, which is competitively antagonized by an IP3-dependent mechanism. TRPC6 channels are also dually regulated by extra- and intra-cellular Ca2+, but potentiative rather than inhibitory effects were far more pronounced. CaM is indispensable for the latter effect.