Resolving the Grip of the Distal Carboxy Tail on the Proximal Calmodulatory Region of CaV Channels

Abstract

Ca2+/calmodulin-dependent inactivation (CDI) of CaV channels plays a crucial role in the homeostasis of intracellular Ca2+, and serves as an ideal prototype for investigating Ca2+ feedback regulation within biological systems. The prevailing view of CDI mechanism is that Ca2+-free calmodulin (apoCaM) preassociates with the IQ domain of the proximal carboxy terminus of channels, and this ‘resident’ calmodulin (CaM) acts as Ca2+ sensor to somehow trigger CDI upon Ca2+ binding. The downstream CaM/channel configurations leading to CDI are complex and under investigation at present. Contrasting with this complexity, CaV1.3 and CaV1.4 feature a long-carboxy-tail splice variant that minimizes CDI by a beautifully simple mechanism. We recently combined electrophysiology and a genetically encoded fluorescent sensor to record CDI and CaM concentration simultaneously (Nature463:968), thereby demonstrating that the distal-carboxy-tail (DCT) harbors an ICDI module that competes with apoCaM for binding to the IQ domain. By kicking off apoCaM, ICDI diminishes CDI in an eminently straightforward manner; thus, the IQ/ICDI complex now looms as a simplified prototype for novel channel modulators, and as an easy entrez into complex CaM/channel configurations underlying CDI. Here, we perform alanine-scanning mutagenesis of the entire IQ domain and ICDI domain. Hotspot mutations which reduce IQ/ICDI binding affinity also commensurately diminish ICDI function (i.e., permit restoration of CDI). Furthermore, the ensemble of effects can be well fit with a Michaelis-Menten equation incorporating the presence of a competitive inhibitor. Thus, the collection of hotspots can be used to identify the minimal functional segments of IQ/ICDI interaction to facilitate structure building, and to provide clues for designing small peptides and/or molecules that regulate CDI of specific CaV channels.