The molecular basis of the inhibition of Ca
| Autor: | Lingjie, Sang, Daiana C O, Vieira, David T, Yue, Manu, Ben-Johny, Ivy E, Dick |
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| Rok vydání: | 2020 |
| Předmět: |
genetic structures
Caveolin 1 PCRD proximal C-terminal regulatory domain ICDI inhibitor of CDI CDI calcium/CaM-dependent inactivation patch clamp FRET fluorescence resonance energy transfer CaV voltage-gated calcium channel DCT distal carboxy tail Structure-Activity Relationship CFP cyan fluorescent protein Calmodulin Humans DCRD distal C-terminal regulatory domain Cells Cultured calcium-dependent inactivation (CDI) CaM calmodulin Ca2+ calcium apoCaM Ca2+ free calmodulin FR fret ratio YFP yellow fluorescent protein Kinetics Mutation ion channel fluorescence resonance energy transfer (FRET) Calcium calcium channel calmodulin (CaM) iTL individually transformed Langmuir Ion Channel Gating Protein Binding Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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