Inhibitory effect of the C-terminal modulator domain of voltage-dependent calcium Cav1.3 channels on the channel gating and calcium-dependent inactivation by single-channel analysis of the native Cav1.3 splice variants

通过对天然 Cav1.3 剪接变体的单通道分析，电压依赖性钙 Cav1.3 通道的 C 端调节域对通道门控和钙依赖性失活的抑制作用

• 批准号: 256584869
• 负责人: Dr. Elza Kuzmenkina
• 金额: --
• 依托单位: Department of Neuroscience
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/256584869?language=en
• 关键词: Inhibitory; effect; terminal; modulator; domain

Due to activation at low potentials, voltage-dependent calcium Cav1.3 channels regulate cellular excitability and pacemaking (e.g., in central neurons and sinoatrial node). On the other hand, calcium entry at low thresholds can lead to calcium overload and result in pathological conditions. For example, the gain of Cav1.3 channel function is reported to be involved in the pathogenesis of Parkinson's and Alzheimer's diseases. The potentially toxic calcium influx through Cav1.3 channels is limited by a negative-feedback process, the calcium-dependent inactivation (CDI). The extent of the channel activity and CDI has to be finetuned for particular cellular needs. The C-terminal modulator (CTM) domain in the channel distal C-terminus shifts channel activation to more depolarized voltages and diminishes apparent CDI. Various CTM splice isoforms are expressed simultaneously in the brain. Whether they have separable functions is not clarified yet. Using a single-channel resolution of the patch-clamp method, we aim at characterizing functional differences among natural Cav1.3 splice variants. The influence of CTM on the different conformational states of the channel can be studied in detail by Markov modeling of the single-channel data. Our second aim is to elucidate the mechanism of CDI inhibition by CTM. It was proposed that intramolecular binding of CTM to the proximal C-terminus competes with the binding of calmodulin, a protein that senses calcium concentration for CDI. However, our preliminary single-channel data suggest that the substantial part of the CDI reduction may arise secondarily: as a result of the inhibition of the channel activation so that the population of states from which CDI occurs is decreased. With CDI modeling, we want to uncouple CTM effects on the channel activation gating and CDI. Further single-channel experiments with calmodulin not binding calcium will provide us with additional information on the channel gating at the fast phase, before CDI occurs. Thus, in this project, we strive to characterize, which regulatory bandwidth and mechanisms are provided by the diversity of CTM splice isoforms in order to shape calcium entry for specific cellular requirements.

由于在低电位下激活，电压依赖性钙Cav1.3通道调节细胞兴奋性和起搏（例如，在中枢神经元和窦房结中）。另一方面，低阈值的钙进入可导致钙超载并导致病理状况。例如，据报道Cav1.3通道功能的获得涉及帕金森病和阿尔茨海默病的发病机制。通过Cav1.3通道的潜在毒性钙内流受到负反馈过程（钙依赖性失活（CDI））的限制。必须针对特定的蜂窝需求对渠道活动和CDI的范围进行微调。通道远端C-末端中的C-末端调制器（CTM）结构域将通道激活转移到更去极化的电压，并减少表观CDI。各种CTM剪接异构体在脑中同时表达。它们是否具有可分离的功能尚未阐明。使用单通道分辨率的膜片钳方法，我们的目的是在自然Cav1.3剪接变异体之间的功能差异的特点。CTM对通道的不同构象状态的影响可以通过单通道数据的马尔可夫建模来详细研究。我们的第二个目的是阐明CTM抑制CDI的机制。有人提出，CTM与近端C-末端的分子内结合与钙调蛋白（一种感测钙浓度的CDI蛋白）的结合竞争。然而，我们初步的单通道数据表明，CDI减少的大部分可能是次要的：作为抑制通道激活的结果，使CDI发生的状态的人口减少。通过CDI建模，我们希望解耦CTM对通道激活门控和CDI的影响。进一步的单通道实验与钙调素不结合钙将为我们提供额外的信息通道门控在快速阶段，CDI发生之前。因此，在本项目中，我们努力表征CTM剪接异构体的多样性提供的调控带宽和机制，以塑造特定细胞需求的钙离子进入。