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

由于低电位激活，电压依赖性钙离子Cav1.3通道调节细胞兴奋性和起搏（如中枢神经元和窦房结）。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，Cav1.3通道功能的获得被报道参与了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的限制，即钙依赖性失活（CDI）。通道活动和CDI的范围必须根据特定的细胞需要进行微调。通道远端c端调制器（CTM）域将通道激活转移到更多的去极化电压，并减少表观CDI。各种CTM剪接异构体在大脑中同时表达。它们是否具有可分离的功能尚不清楚。利用膜片钳方法的单通道分辨率，我们旨在表征天然Cav1.3剪接变体之间的功能差异。CTM对通道不同构象状态的影响可以通过对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中药抑制CDI的机制。有人提出，CTM与近端c端分子内结合与钙调蛋白的结合竞争，钙调蛋白是CDI感知钙浓度的蛋白质。然而，我们的初步单通道数据表明，CDI减少的实质性部分可能是次要的：由于通道激活受到抑制，因此CDI发生的状态数量减少。通过CDI建模，我们想要解耦CTM对通道激活门控和CDI的影响。钙调素不结合钙的进一步单通道实验将为我们提供CDI发生前快速阶段通道门控的额外信息。因此，在本项目中，我们努力表征CTM剪接异构体多样性提供的调节带宽和机制，以形成特定细胞需求的钙进入。

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.