Role of cytoskeletal elements in calcium channel trafficking and function

钙离子对生理功能的各个方面都很重要，包括记忆、神经信号的传输和心跳的产生。钙进入细胞是由钙通道介导的，钙通道是嵌入细胞膜的一系列蛋白质，允许钙离子通过细胞膜。由于过高的钙水平可能对细胞有害，因此通过这些渠道进入的钙量受到严格控制。因此，考虑到它们的关键作用，了解钙通道如何在分子水平上发挥作用，以及细胞如何调节存在的钙通道蛋白的量是至关重要的。在钙通道家族中，有一个亚类被称为T型钙通道。它们对于调节神经细胞的电兴奋性以及某些类型的激素和神经递质的释放特别重要。在这一应用中，我们建议研究控制T型钙通道从细胞内部合成和组装到细胞膜上调节钙内流的特定位置的分子基础。我们已经获得的初步数据显示，T型钙通道与幽灵蛋白相互作用并受其控制，幽灵蛋白是构成细胞内部骨架的一种重要蛋白质。我们现在将研究T型通道是如何与血影蛋白和相关的蛋白ankyrin相互作用的，以及这些相互作用如何调节1)通道到细胞膜的运输，2)通道在细胞膜中的流动性，以及3)通道的功能。我们将结合使用最先进的实验技术来实现我们的目标。这些包括分子生物学、生物化学、模型系统和活神经细胞中钙通道的电子记录，以及使用复杂的成像技术实时跟踪通道的运动。具体地说，我们将通过DNA技术操纵钙通道及其调节蛋白的行为，然后研究这如何影响它们的组装、运输和膜的稳定性。最后，我们将确定神经细胞电活动的后果。总而言之，这项工作将为一个适用于广泛物种的基本生物过程提供新的见解。

Calcium ions are important for a variety of aspects of physiological function including memory, transmission of nerve signals, and the generation of the heart beat. Calcium entry into cells is mediated by calcium channels, a family of proteins which are embedded in the cell membrane and allow calcium ions to pass through them. Because excessive calcium levels can be toxic to cells, the amount of calcium entering through these channels is tightly regulated. Given their pivotal roles, it is therefore critical to understand how the calcium channels function at the molecular level, and how cells regulate the amount of calcium channel protein that is present. Among the family of calcium channels is a subclass that is called T-type calcium channels. These are particularly important for regulating the electrical excitability of nerve cells and for the release of certain types of hormones and neurotransmitters. In this application, we propose to investigate the molecular basis that governs the trafficking of T-type calcium channels from the inside of the cells where they are synthesized and assembled, to specific loci in the cell membrane where they regulate calcium entry. We have obtained preliminary data revealing that T-type calcium channels interact with and are controlled by spectrin, an important protein that forms part of the cell’s internal skeleton. We will now investigate how T-type channels interact with spectrin and the associated protein ankyrin, and how these interactions regulate 1) the trafficking of the channels to the cell membrane, 2) the mobility of the channels in the cell membrane, and 3) the function of the channels. We will use a combination of state of the art experimental techniques to tackle our objectives. These include molecular biology, biochemistry, electrical recordings from calcium channels in model systems and in living nerve cells, and the use of sophisticated imaging techniques to track the movement of the channels in real time. Specifically, we will manipulate the behavior of calcium channels and their regulatory proteins via DNA technology, and then examine how this affects their assembly, trafficking, and membrane stability. Finally, we will determine the consequences for the electrical activity of nerve cells. Altogether, this work will provide novel insights about a fundamental biological process that is applicable across a wide range of species.