Inhibition by Reduction of PIP2 Accelerates Inactivation of Receptor-Operated TRPC6/7 Currents

TRPC3, C6 and C7, which comprise a subfamily of the mammalian diacylglycerol (DAG)-activated TRPC cation channels, play key roles in cardiovascular, gastrointestinal, renal and nervous system physiology. We recently reported that the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) dominantly inhibits these TRPC channels activity, irrespective of the presence of a DAG analogue. However, the functional significance of PI(4,5)P2 reduction/depletion during G(q) protein-coupled receptor-operated TRPC activation remains largely unknown. To address this question, we simultaneously measured TRPC current kinetics and PI(4,5)P2/DAG dynamics using Forster resonance energy transfer (FRET) at varying degrees of receptor stimulation. Our measurements demonstrated a clear correlation between the time-course of activation/inactivation in TRPC6/7 currents and the kinetics of PI(4,5)P2 reduction. In contrast, the time-course of DAG production is incompatible with TRPC6/7 current inactivation, indicating that reduction of PI(4,5)P2 is primarily responsible for this inhibition. After estimating the functional dissociation constant of PI(4,5)P2 binding to these DAG-sensitive TRPC channels, we used a model simulation to demonstrate the similarity of receptor-operated TRPC6/7 currents in both a HEK293 cell recombinant expression system and aortic smooth muscle cells (A7r5). Our findings demonstrate that G(q) protein-coupled receptor stimulation causes a reduction in PI(4,5)P2 levels that inhibits TRPC6/7 channels by accelerating their inactivation and perhaps limiting their maximum open probability.