The molecular basis of the inhibition of Ca(V)1 calcium-dependent inactivation by the distal carboxy tail

Ca(2+)/calmodulin-dependent inactivation (CDI) of Ca(V) channels is a critical regulatory process that tunes the kinetics of Ca(2+) 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(V)1.3 and Ca(V)1.4 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(V)1.3 IQ domain and the Ca(V)1.4 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(V)1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate Ca(V)1.2 CDI. Yet coexpression of Ca(V)1.2 ICDI with select Ca(V)1.3 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 Ca(2+)-entry and supports normal neuronal and cardiac function.