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

描述（由申请人提供）：钙离子反转运蛋白（CaCA）家族对控制细胞内钙信号传导很重要。Ca2+稳态的维持依赖于调节Ca2+的特定运输系统的功能。该家族的一个成员，心脏肌层Na+-Ca2+交换器（NCX1），是Ca2+从肌细胞外排的主要机制。因此，NCX1密切参与心肌松弛和防止细胞Ca2+超载。NCX1也与一些心律失常的发生有关，并在心力衰竭中增加功能。这项研究将产生CaCA交换物家族中医学相关蛋白NCX1的机制细节。此外，我们将为CaCA家族的表达、纯化、结晶和物相测定开发新的方法。这项技术将直接转移到其他膜蛋白家族，以产生高分辨率的结构。特别是，我们的目标是实现NCX1的细胞内调节环的3D结构，NCX1是哺乳动物心肌细胞Ca2+挤出的主要调节剂，以及来自CaCA家族原核成员的完整交换器的结构。结构分析的所有假设都将通过功能分析进行检验。从这些结构/功能研究中获得的知识可能会导致合理的药物设计，并解决这种心脏和神经生理学关键调节剂功能的剩余未知方面。公共卫生相关性：CaCA蛋白家族是普遍存在的Ca2+信号通路的重要组成部分，用于在各种细胞类型中维持Ca2+稳态。哺乳动物Na+-Ca2+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Ca2+过载密切相关。确定CaCA家族成员的结构和调节功能，特别是NCX1，将允许合理设计可能用于调节健康和疾病中与功能失调的Ca2+运输相关的心脏收缩性的新药。

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.