Factors regulating Cav 2.1 modulation by Ca2+ in neurons

描述(申请人提供)：电压门控钙离子通道将膜去极化与钙离子流入细胞相结合，启动了广泛的重要生物学过程，包括磷酸化、基因转录和神经递质释放。由于这些钙离子通道对细胞信号的重要性，它们的活性受到多种形式的调节。特别是，Cav2.1(P/Q型)钙通道经历了钙离子的双重反馈调节，这有助于某些突触的短期可塑性。在重复刺激中，Cav2.1钙电流最初增加(促进)，然后逐渐减少(失活)。易化和失活都依赖于钙离子，因此可以受到控制细胞内钙离子水平的因素的影响。这项研究的初步结果表明，一些神经元中高浓度存在的钙缓冲蛋白和细胞内储存的钙离子可能是钙离子调节Cav2.1的重要决定因素。研究计划中概述的实验将检验这一假设，即这些因素对神经元Cav2.1通道受钙离子调制的程度产生关键影响。全细胞膜片钳电生理学、分子生物学和钙成像技术将被用来表征在转基因细胞和分离的神经元中，钙缓冲蛋白和细胞内钙库如何影响钙离子对Cav2.1活性依赖的反馈。这项研究的发现可能揭示神经元中Cav2.1通道异质性的新机制，并为替代治疗与钙通道缺陷相关的疾病(如偏头痛、癫痫和共济失调)提供新的见解。

DESCRIPTION (provided by applicant): Voltage-gated Ca2+ channels couple membrane depolarization to the influx of Ca2+ into the cell, which initiates a wide variety of biologically important processes including phosphorylation, gene transcription and neurotransmitter release. Because of the importance of these Ca2+ channels for cellular signaling, their activity is subject to numerous forms of regulation. In particular, Cav2.1 (P/Q-type) Ca2+ channels undergo a dual feedback regulation by Ca2+ ions which contributes to short-term plasticity at some synapses. During repetitive stimuli, Cav2.1 Ca2+ currents initially increase (facilitate) and gradually decrease (inactivate). Both facilitation and inactivation are Ca2+-dependent and therefore can be influenced by factors controlling intracellular Ca2+ levels in neurons. Preliminary results presented in this proposal indicate that Ca2+ buffering proteins, which are present at high concentrations in some neurons, and Ca2+ in intracellular stores may be important determinants of Cav2.1 regulation by Ca2+. The experiments outlined in the research plan will test the hypothesis that these factors critically influence the extent to which neuronal Cav2.1 channels are modulated by Ca2+. Whole-cell patch clamp electrophysiology, molecular biology, and Ca2+ imaging techniques will be used to characterize how Ca2+ buffering proteins and intracellular Ca2+ stores affect activity-dependent feedback of Cav2.1 by Ca2+ both in transfected cells and isolated neurons. The findings from this research may reveal novel mechanisms underlying the heterogeneous properties of Cav2.1 channels in neurons and provide new insights into alternative strategies to treat diseases associated with Ca2+ channel defects, such as migraine, epilepsy and ataxia.