Full-length transient receptor potential (TRP) cation channel TRPC4␣ and shorter TRPC4 lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca
2؉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␣ but not TRPC4 was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP 2 ). 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␣ indicating that PIP 2 inhibits TRPC4␣ in a highly selective manner. We show that PIP 2 binds to the C terminus of TRPC4␣ but not that of TRPC4 in vitro. Its inhibitory action was dependent on the association of TRPC4␣ with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-ThrArg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP 2 breakdown appears to be a required step in TRPC4␣ channel activation as PIP 2 depletion alone was insufficient for channel opening, which additionally required Ca 2؉ and pertussis toxin-sensitive G i/o proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP 2 /cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonistactivated cation channels in ileal myocytes.