Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate

Abstract

Full-length transient receptor potential (TRP) cation channel TRPC4α and shorter TRPC4β 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 Gq/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4α but not TRPC4β was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast, several other phosphoinositides (PI), including PI(3,4)P2, PI(3,5)P 2, and PI(3,4,5)P3, had no effect or even potentiated TRPC4α indicating that PIP2 inhibits TRPC4α in a highly selective manner. We show that PIP2 binds to the C terminus of TRPC4α but not that of TRPC4β in vitro. Its inhibitory action was dependent on the association of TRPC4α 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 TRPC4α channel activation as PIP 2 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.