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

描述（由申请人提供）：钙-阳离子逆向转运蛋白（CaCA）家族对于控制细胞内钙信号传导非常重要。Ca 2+稳态的维持依赖于调节Ca 2+的特定转运系统的功能。该家族的一个成员，心肌肌膜Na+-Ca 2+交换器（NCX 1），是Ca 2+从肌细胞流出的主要机制。因此，NCX 1密切参与心肌松弛和防止细胞Ca 2+超载。NCX 1也与一些心律失常的发生有关，并且在心力衰竭中具有增加的功能。这项研究将产生一个医学相关的蛋白质从CaCA家庭的交换器，NCX 1的机制细节。此外，我们将开发新的方法表达，纯化，结晶和相确定的CaCA家族。这项技术将直接转移到其他家族的膜蛋白，以产生高分辨率的结构。特别是，我们的目标是实现NCX 1的细胞内调节环的3D结构，NCX 1是哺乳动物心肌细胞中Ca 2+挤出的主要调节因子，以及CaCA家族原核成员的完整交换器的结构。将通过功能试验对结构分析的所有假设进行检验。从这些结构/功能研究的知识可能会导致合理的药物设计和解决剩余的未知方面的功能，心脏和神经生理学的这一关键调节。公共卫生相关性：CaCA蛋白家族是用于维持多种细胞类型中Ca 2+稳态的Ca 2+信号传导途径的普遍存在和重要组分。哺乳动物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Ca 2+超载密切相关。确定CaCA家族成员的结构和调节功能，特别是NCX 1，将允许合理设计可用于调节与功能失调的Ca 2+转运相关的健康和疾病中的心肌收缩力的新型药物。

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.