A Self-Limiting Regulation of TRPC3 C6 C7 Channels Linked with PI(4,5)P2-Diacylglycerol Signaling

TRPC3/C6/C7 channels are activated by diacylglycerol (DAG) upon the phospholipase C (PLC) coupled receptor stimulation which results in the breakdown of phosphoinositides (PIPs). Although the critical function of PIPs on various ion transporting molecules has been demonstrated, their roles in TRPC3/C6/C7 channels remain controversial. Here, by ectopical usage of voltage-sensing PIPs phosphatase (DrVSP), we found that dephosphorylation of PIPs produces robust inhibition of currents induced by carbachol (CCh), OAG and RHC80267, for all TRPC3/C6/C7 channels. The strength of inhibition was, however, variable with the rank order of DrVSP-mediated inhibition (VMI): C3 < C6 < C7. Pharmacological and molecular interventions most likely suggest that depletion of PI(4,5)P2 is a critical event for VMI of all three channels. As predicted, when PLC catalytic signal was vigorously activated through overexpressed muscarinic type-I receptor (M1R), macroscopic kinetics of TRPC currents were greatly accelerated as similar the rank order of VMI, and it was simultaneously dismissed or attenuated. Likewise, VMI was rarely detected in vasopressin-induced TRPC6-like currents from smooth muscle cells (A7r5), supporting a severe or various degree of PI(4,5)P2 depletion in the physiological conditions. We also employed simultaneous detection of FRET-based measurement of PI(4,5)P2 and TRPC6 current, confirming that VMI reflects the degree of PI(4,5)P2 depletion. These results thus demonstrate that TRPC3/C6/C7 channels are differentially regulate by depletion of PI(4,5)P2, and bimodal signal produced by PLC activation (i.e. depletion of PIP2 and production of DAG) simultaniously controls these channels in a self-limiting manner.