PLC-mediated PI(4,5)P₂ hydrolysis regulates activation and inactivation of TRPC6/7 channels

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) by phospholipase C (PLC). PI(4,5)P₂ depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P₂ reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P₂or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to G_q and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P₂ reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P₂. We determined the functional dissociation constant of PI(4,5)P₂ to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P₂, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P₂ and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P₂ regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P₂ depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P₂ in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.