Isoform-specific Inhibition of TRPC4 Channel by Phosphatidylinositol 4,5-Bisphosphate

Full-length transient receptor potential (TRP) cation channel TRPC4 alpha and shorter TRPC4 beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca2+ influx. In common with other classical TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4 alpha but not TRPC4 beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast, several other phosphoinositides (PI), including PI(3,4)P-2, PI(3,5)P-2, and PI(3,4,5)P-3, had no effect or even potentiated TRPC4 alpha indicating that PIP2 inhibits TRPC4 alpha in a highly selective manner. We show that PIP2 binds to the C terminus of TRPC4 & but not that of TRPC4 beta in vitro. Its inhibitory action was dependent on the association of TRPC4 alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP2 breakdown appears to be a required step in TRPC4 alpha channel activation as PIP2 depletion alone was insufficient for channel opening, which additionally required Ca2+ and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP2/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist activated cation channels in ileal myocytes.