Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
J Biol Chem. 2021 Jan-Jun;296:100502. doi: 10.1016/j.jbc.2021.100502. Epub 2021 Mar 2.
Ca/calmodulin-dependent inactivation (CDI) of Ca channels is a critical regulatory process that tunes the kinetics of Ca entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the Ca carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of Ca1.3 and Ca1.4 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 Ca1.3 IQ domain and the Ca1.4 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, Ca1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate Ca1.2 CDI. Yet coexpression of Ca1.2 ICDI with select Ca1.3 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-entry and supports normal neuronal and cardiac function.
钙/钙调蛋白依赖性失活（CDI）是调节不同细胞类型和生理反应中钙内流动力学的关键调节过程。CDI 是由钙调蛋白（CaM）介导的，它与 Ca 羧基尾部的 IQ 结构域结合。这种调节过程通过选择性剪接进行调节，使得 Ca1.3 和 Ca1.4 的某些剪接变体包含长的远端羧基尾部（DCT）。DCT 具有 CDI 抑制剂（ICDI）模块，该模块竞争性地将 CaM 从 IQ 结构域置换出来，从而减弱 CDI。虽然这个总体机制现在已经描述得很清楚，但 ICDI 与 IQ 结构域结合所需的详细相互作用仍有待阐明。在这里，我们对 IQ 和 ICDI 结构域进行丙氨酸扫描突变，并评估邻近区域对 CDI 抑制的贡献。通过 FRET 结合分析，我们确定了 Ca1.3 IQ 结构域和 Ca1.4 ICDI 及附近 A 区域内的功能相关残基，这些残基对于高亲和力 IQ/ICDI 结合是必需的。重要的是，膜片钳记录表明，这种相互作用的破坏会相应地减弱 ICDI 的功能，导致突变通道中 CDI 的重新出现。此外，Ca1.2 通道具有同源的 DCT；然而，该通道的 ICDI 区域似乎不会明显调节 Ca1.2 CDI。然而，Ca1.2 ICDI 与某些 Ca1.3 剪接变体的共表达显著破坏了 CDI，这表明在表达这两种通道亚型的细胞中存在跨通道调节方案。总之，这些发现为微调钙内流并支持正常神经元和心脏功能的分子变阻器提供了新的见解。
