Na and Ca Transport Across the Plasma Membrane of Squid Giant Axons: Mechanisms of Metabolic Control of the Na/Ca Exchanger

钠/钙交换器是负责调节细胞内钙浓度的细胞膜蛋白。它作为反向转运，从不同的膜侧将一个钙离子（两个正电荷）交换为三个钠离子（三个正电荷）;即它是产电的，因为转运电荷的不平衡产生电流。钙逆梯度转运的能量取自钠离子的梯度加上跨膜的膜电位差。除了钠-钙竞争转运位点外，该机制还显示了细胞内和细胞外配体的调节。在细胞内侧有两种主要类型的调节：（1）离子：钠失活、钙刺激和质子抑制，以及（2）代谢：ATP和磷酸精氨酸（PA）刺激。ATP的调节需要镁和低分子量的可溶性胞质调节蛋白（SCRP）; PA需要镁，但不需要SCRP。在拟议的工作中，研究人员旨在通过使用两种实验制剂来个性化和表征鱿鱼钠/钙交换器的离子和代谢调节之间的相互关系：（i）内部透析下的鱿鱼轴突，一种允许对细胞内和细胞外环境进行离子和生物化学控制的基本上“体内”制备，和（ii）鱿鱼神经膜囊泡，其允许进行平行转运和生物化学测定。该计划包括：（i）研究ATP、PA、钠和质子之间的联系以及细胞内镁在钠/钙交换调节中的相关性;（ii）用32 P标记的PA研究是否有膜磷酸化参与PA刺激。(iii)目的分离和鉴定低分子量可溶性胞质调节蛋白（SCRP）。必须强调预期结果的广泛影响：首先，提供对心血管系统和大脑正常功能的见解。第二，帮助了解疾病，如心脏和大脑缺氧和缺血，和高血压，情况下，是一个大的不平衡的配体影响最重要的膜系统之一的细胞钙调节在大脑和心血管系统，钠/钙交换器。

The Sodium/Calcium exchanger is an integral cell membrane protein in charge of regulation of intracellular Calcium concentration. It works as a counter-transport, exchanging one Calcium ion (two positive charges) for three Sodium ions (three positive charges) from different membrane sides; i.e. it is electrogenic because the unbalance of transported charges generates current. The energy for Calcium transport against a gradient is taken from the gradient of Sodium ions plus the electrical membrane potential difference across the membrane. In addition to Sodium-Calcium competition for the transporting sites, this mechanism displays regulation by intra- and extracellular ligands. There are two main types of regulation on the intracellular side: (1) Ionic: Sodium inactivation, Calcium stimulation and Proton inhibition, and (2) Metabolic: ATP and phosphoarginine (PA) stimulation. Regulation by ATP requires Magnesium and also a low molecular weight soluble cytosolic regulatory protein (SCRP); PA needs Magnesium but no SCRP The metabolic pathways for ATP and PA are different. In the proposed work the investigator aims to individualize and characterize the interrelationships between ionic and metabolic regulation of the squid Sodium/Calcium exchanger by using two experimental preparations: (i) Squid axons under internal dialysis, an essentially "in vivo" preparation that permits ionic and biochemical control of intra- and extracellular environments, and (ii) Squid nerve membrane vesicles, that allow to perform parallel transport and biochemical assays. The plan includes: (i) To study the links between ATP, PA, Sodium and Protons and the relevance of the intracellular Magnesium in the modulation of the Sodium/Calcium exchanger; (ii) To use 32P-labeled PA to investigate if there are membrane phosphorylations involved in the PA stimulation. (iii) To isolate and identify the low molecular weight soluble cytosolic regulatory protein (SCRP) essential for ATP regulation. It must be emphasized the broad impact of the expected results: First, provide insights on normal function of the cardiovascular system and brain. Second, help to understand diseases, like cardiac and brain hypoxia and ischemia, and hypertension, situations where the is a large imbalance of the ligands affecting one of the most important membrane system for cell Calcium regulation in brain and cardiovascular system, the Sodium/Calcium exchanger.