Structure/Function Studies on the Ca2+: Cation Antiporter family of transporters

描述(申请人提供)：钙离子反向转运蛋白(CACA)家族的蛋白质对于细胞内钙信号的控制很重要。钙离子稳态的维持依赖于调节钙离子的特定转运系统的功能。心肌细胞膜Na+-Ca~(2+)交换器(NCX1)是该家族的成员之一，是心肌细胞内钙外流的主要机制。因此，NCX1与心肌松弛和防止细胞内钙超载密切相关。NCX1还与一些心律失常的发生有关，并增加了心力衰竭的功能。这项研究将产生来自CacA交换器家族NCX1的一种与医学相关的蛋白质的机制细节。此外，我们还将为CACA家族的表达、纯化、结晶和相态确定开发新的方法学。这项技术将直接转移到其他膜蛋白家族，以产生高分辨率结构。特别是，我们的目标是获得NCX1细胞内调控环的3D结构，NCX1是哺乳动物心肌细胞钙排出的主要调节因子，以及CACA家族原核成员的完整交换器的结构。结构分析中的所有假设都将通过功能分析进行检验。来自这些结构/功能研究的知识可能导致合理的药物设计，并解决这一心脏和神经生理学的关键调节因子功能的剩余未知方面。与公共健康相关：CACA蛋白家族是钙信号通路中无处不在的重要组成部分，用于维持各种细胞类型的钙稳态。哺乳动物的Na+-Ca~(2+)交换器(NCX)是该家族中最具特征的成员，与心肌松弛和防止细胞内钙超载密切相关。确定CACA家族成员，特别是NCX1的结构和调节功能，将允许合理设计新的药物，这些药物可能用于调节健康和与钙转运功能障碍相关的疾病的心脏收缩性能。

DESCRIPTION (provided by applicant): The calcium-cation antiporter (CaCA) family of proteins is important for the control of intracellular calcium signaling. Maintenance of Ca2+ homeostasis depends on the functioning of specific transport systems that regulate Ca2+. One member of this family, the cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1), is the primary mechanism for Ca2+ efflux from myocytes. As such, NCX1 is intimately involved in cardiac muscle relaxation and in preventing cellular Ca2+ overload. NCX1 has also been implicated in the genesis of some cardiac arrhythmias and has increased function in heart failure. This study will generate mechanistic details of a medically relevant protein from the CaCA family of exchangers, NCX1. In addition, we will develop new methodology for the expression, purification, crystallization and phase determination of the CaCA family. This technology will be directly transferable to other families of membrane proteins to yield high-resolution structures. In particular, we aim to achieve a 3D structure of the intracellular regulatory loop of NCX1, the primary regulator of Ca2+ extrusion from mammalian cardiac myocytes, as well as the structure of a complete exchanger from a prokaryotic member of the CaCA family. All hypotheses from the structural analysis will be tested by functional assays. The knowledge from these structure/function studies may lead to rational drug design and to the resolution of remaining unknown aspects of the function of this critical regulator of cardiac and neural physiology. PUBLIC HEALTH RELEVANCE: The CaCA family of proteins are ubiquitous and vital components of Ca2+ signaling pathways utilized for maintaining Ca2+ homeostasis in a variety of cell types. The mammalian Na+-Ca2+ exchanger (NCX), the best- characterized member of the family, is intimately involved in cardiac muscle relaxation and in preventing cellular Ca2+ overload. Determining the structure and regulatory function of CaCA family members in general, and NCX1 in particular, will allow for rational design of novel pharmaceuticals that may be used in regulating cardiac contractility in health and disease related to dysfunctional Ca2+ transport.