Resolving the molecular fingerprint of the distal carboxy tail in modulating CaV1 calcium dependent inactivation

Ca2+/calmodulin-dependent inactivation (CDI) of CaV channels is a critical regulatory process required for tuning the kinetics of Ca2+ entry for different cell types and physiologic responses. Calmodulin (CaM) resides on the IQ domain of the CaV carboxy-tail, such that Ca2+ binding initiates a reduction in channel open probability, manifesting as CDI. This regulatory process exerts a significant impact on Ca2+ entry and is tailored by alternative splicing. CaV1.3 and CaV1.4 feature a long-carboxy-tail splice variant that modulates CDI through a competitive mechanism. In these channels, the distal-carboxy-tail (DCT) harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well-described, the detailed interaction loci for ICDI binding to the IQ domain is yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions. We identify multiple critical residues within the IQ domain, ICDI and the nearby A region of the channel, which are required for high affinity IQ/ICDI binding. Importantly, disruption of this interaction commensurately diminishes ICDI function, as seen by the re-emergence of CDI in mutant channels. Furthermore, analysis of the homologous ICDI region of CaV1.2 reveals a selective effect of this channel region on CaV1.3 channels, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine tunes Ca2+ entry and supports normal neuronal and cardiac function.