Departure of Calmodulin from the IQ Domain of Ca V1.3 Channels During Calcium-Dependent Inactivation

Abstract

Studies of calmodulin (CaM) regulation of CaV channels have long focused on an IQ domain in the carboxy-terminus of channels. Strong evidence indicates that Ca2+-free CaM (apoCaM) preassociates with the IQ domain before Ca2+ entry through channels (e.g., Liu et alNature 463:968), and that Ca2+/CaM has the potential to bind the IQ. Hence, Ca2+-dependent inactivation (CDI) of channels has been proposed to result from Ca2+-dependent conformational changes of CaM, all while bound to the IQ. Yet, analysis of Ca2+/CaM complexed with the IQ domain of CaV2.1 (Structure 16:607) hints that Ca2+/CaM departs from the IQ domain during channel regulation. To generalize this hypothesis, we here alanine scanned the entire IQ domain of CaV1.3, a prototype channel homologous to long-studied CaV1.2. Surprisingly, CDI was strongly diminished at only three residues: the signature isoleucine (I[0]), an alanine situated four residues upstream (A[-4]), and a downstream phenylalanine (F[4]). How do these mutations decrease CDI? If CDI were suppressed by inhibiting Ca2+/CaM binding to an IQ effector site, then Ca2+/CaM binding to this domain should exhibit commensurate disruption. However, FRET two-hybrid assays indicated no such decrease in binding. Alternatively, mutations could attenuate CDI by inhibiting apoCaM preassociation with the channel. Indeed, binding assays revealed strong mutational effects on apoCaM interaction with the IQ domain, consistent with the observed CDI deficits. Indeed, overexpression of wild-type CaM largely restored CDI of mutant channels, fitting with recovery of apoCaM preassociation via mass action. Thus, while the IQ domain appears to be important as a preassociation locus for apoCaM, CDI is likely triggered by the departure of Ca2+/CaM to interact with effector sites elsewhere on the channel (Ben Johny, Yang, and Yue, this meeting). This departure would fundamentally transform our understanding of CaM/channel regulation.