The molecular basis of the inhibition of Ca_V1 calcium-dependent inactivation by the distal carboxy tail

Ca²⁺/calmodulin-dependent inactivation (CDI) of Ca_V channels is a critical regulatory process that tunes the kinetics of Ca²⁺ entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the Ca_V carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of Ca_V1.3 and Ca_V1.4 contain a long distal carboxy tail (DCT). The 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 interactions required for ICDI binding to the IQ domain are yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions to CDI inhibition. Through FRET binding analysis, we identify functionally relevant residues within the Ca_V1.3 IQ domain and the Ca_V1.4 ICDI and nearby A region, which are required for high-affinity IQ/ICDI binding. Importantly, patch-clamp recordings demonstrate that disruption of this interaction commensurately diminishes ICDI function resulting in the re-emergence of CDI in mutant channels. Furthermore, Ca_V1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate Ca_V1.2 CDI. Yet coexpression of Ca_V1.2 ICDI with select Ca_V1.3 splice variants significantly disrupts CDI, 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.