Regulation of L-type calcium channel inactivation

钙离子通过由膜电压变化激活的钙通道进入大脑中的神经元，这一过程对从基因转录到膜兴奋性的功能至关重要。事实上，钙作为信号分子的重要性决定了需要系统来精确调节钙进入的时间和程度。钙通道的“L型”（Cav 1）类在大脑中广泛表达，并受钙调蛋白（CaM）调节，钙调蛋白是一种通过“钙依赖性失活”（CDI）或“钙依赖性促进”（CDF）过程修饰Cav 1钙内流的分子。 最近的研究表明，Cav 1介导的钙内流可以通过与通道C末端结合以减少CDI的辅助蛋白质而显著增加。 两个例子是支架蛋白致密素，其结合到C末端以募集专门的激酶CaMKII，第二个是钙敏感蛋白钙调素，其物理地取代Cav 1通道上的CaM。我们有一种新形式的Cav1.3通道易化的初步数据，这种通道易化在电路活动的一系列输入特征之后持续>5秒（称为L-CDF）。此外，Cav1.3 L-CDF依赖于densin和CaMK II的表达。 这一点很重要，因为这些蛋白质中的每一种都在CA 1海马锥体细胞中表达，其中在短暂的突触输入序列后钙通道活性的“延迟促进”增强了钙内流。我们推测，辅助蛋白densin和caldendrin的基础上延迟易化，促进了大量增加的钙内流在锥体细胞。我们将应用从分子生物学到生物化学到电生理学的技术来研究在模型系统和啮齿动物脑组织切片中表达的L-型钙通道，以确定致密蛋白和钙枝蛋白在产生Cav 1通道L-CDF中的作用。这项工作代表了我们实现长期目标的第一步， 了解L型钙通道是如何调节的， 内部钙和第二信使途径的激活。Cav 1通道在从大脑到肌肉和心脏的所有组织中的广泛表达将确保结果对于理解钙内流在多种情况下如何控制非常重要，从而推进了体内可兴奋细胞的功能如何通过这些蛋白质-蛋白质相互作用进行调节的知识。

Calcium ions enter neurons in the brain through calcium channels activated by a change in membrane voltage, a process that is critical to functions ranging from gene transcription to membrane excitability. Indeed, the importance of calcium as a signaling molecule dictates the need for systems to precisely regulate the timing and extent of calcium entry. The “L-type” (Cav1) class of calcium channels is widely expressed in the brain and is regulated by calmodulin (CaM), a molecule that modifies Cav1 calcium influx through a process of “calcium-dependent inactivation” (CDI) or “calcium-dependent facilitation” (CDF). Recent work reveals that Cav1-mediated calcium influx can be substantially increased by accessory proteins that bind to the channel C terminus to reduce CDI. Two examples are the scaffolding protein densin that binds to the C terminus to recruit the specialized kinase CaMKII, and a second is the calcium sensitive protein caldendrin that physically displaces CaM on the Cav1 channel. We have preliminary data for a novel form of Cav1.3 channel facilitation that lasts for >5 seconds (termed L-CDF) following a brief train of input characteristic of circuit activity. Moreover, Cav1.3 L-CDF depends on expression of densin and CaMKII. This is important, as each of these proteins are expressed in CA1 hippocampal pyramidal cells, where a “delayed facilitation” of calcium channel activity after a brief train of synaptic input enhances calcium influx. We hypothesize that the accessory proteins densin and caldendrin underlie the delayed facilitation that promotes a large increase in calcium influx in pyramidal cells. We will apply techniques ranging from molecular biology to biochemistry to electrophysiology to study L-type calcium channels expressed in a model system and in slices of rodent brain tissue to determine the role of densin and caldendrin in producing Cav1 channel L-CDF. Together this work represents the first step of attaining our long term goal of understanding how L-type calcium channels are regulated to control internal calcium and activation of second messenger pathways. The widespread expression of Cav1 channels in all tissues ranging from brain to muscle and heart will ensure that the results will be immediately important to understanding how calcium influx is controlled in multiple contexts, advancing knowledge of how the function of excitable cells in the body is regulated by these protein-protein interactions.