Molecular and Structural Basis of Dual Regulation of a Canonical TRP Channel by Calmodulin

Abstract

The canonical transient receptor potential (TRPC) channels are widely distributed and have diverse biological functions. They are activated by stimulation of phospholipase C-coupled receptors, resulting in membrane depolarization and Ca2+ influx, which in turn feedback to regulate the channel activity through the Ca2+-binding protein calmodulin (CaM) and other signaling pathways. Previous biochemical studies indicate that TRPC subunits contain one to four putative CaM-binding sites. One of these sites is named the “CaM-IP3receptor binding” or CIRB site (because it also interacts with an IP3 receptor fragment in vitro). The CIRB site is conserved in all seven TRPC subunits. CaM exerts either stimulatory or inhibitory effects on different TRPC channels. However, the molecular mechanism of CaM modulation of TRPC channels is unclear. We have solved the crystal structure of the complex of CaM and the CIRB site of TRPC5 channels, which regulate growth cone morphology and neurite growth, and require CaM for agonist-induced activation. The structure shows that the two lobes of a single Ca2+-bound CaM (Ca2+/CaM ) bind two CIRB peptides arranged in parallel. This peptide dimerizes only in the presence of Ca2+/CaM, suggesting that Ca2+/CaM binding to the CIRB site may induce major conformational changes in intact channels. Structure-based mutagenesis studies show that Ca2+/CaM binding to the CIRB site is not required for agonist-induced channel activation, but it safeguards the channel against inhibition produced by CaM binding to another site on the channel. We have identified this inhibitory site and found it to be a novel CaM-binding motif that can interact with not only Ca2+/CaM but also CaM1234, a mutant CaM deficient in binding Ca2+. Our results provide new insights into the intricate feedback regulation of a canonical TRP channel.