The molecular basis of the inhibition of CaV1 calcium-dependent inactivation by the distal carboxy tail.
Ca2+/calmodulin-dependent inactivation (CDI) of CaV 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 CaV carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of CaV1.3 and CaV1.4 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 CaV1.3 IQ domain and the CaV1.4 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, CaV1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate CaV1.2 CDI. Yet coexpression of CaV1.2 ICDI with select CaV1.3 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.
['Sang|Lingjie|L|', 'Vieira|Daiana C O|DCO|', 'Yue|David T|DT|', 'Ben-Johny|Manu|M|', 'Dick|Ivy E|IE|']
D002118:Calcium D002147:Calmodulin D051242:Caveolin 1 D002478:Cells, Cultured D006801:Humans D015640:Ion Channel Gating D007700:Kinetics D009154:Mutation D011485:Protein Binding D013329:Structure-Activity Relationship
calmodulin (CaM) calcium channel calcium-dependent inactivation (CDI) fluorescence resonance energy transfer (FRET) ion channel patch clamp