The molecular basis of the inhibition of Ca1 calcium dependent inactivation by the distal carboxy tail
JournalJournal of Biological Chemistry
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AbstractCa2+/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 co-expression 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.
Rights/TermsCopyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
calcium dependent inactivation (CDI)
fluorescence resonance energy transfer (FRET)
Identifier to cite or link to this itemhttp://hdl.handle.net/10713/15561
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