Transport and Biochemical Events Associated With Metabolic Regulation of the Squid Nerve Na+/Ca2+ Exchanger

钠/钙反向转运是一种普遍存在的结构，其可逆地将钠交换为钙穿过细胞膜。该系统受细胞内离子和代谢的显著调节。在离子中，钠和质子是抑制性的，而钙是刺激性的。关于代谢，ATP拮抗离子抑制，而在鱿鱼神经中，磷酸精氨酸（PA）通过促进钙与其转运位点的结合来刺激。ATP的刺激需要低分子量的可溶性胞质调节蛋白（SCRP）; PA的刺激似乎与另一种膜结合蛋白小神经丝有关。离子和ATP调节发生在交换蛋白的大的细胞内区域。相反，PA调制可能发生在其他未指明的位置。实验证据表明，与ATP和PA调节有关的化学反应是不同的。然而，研究人员最近发现，ATP可以防止细胞酸化（质子浓度增加）后PA调节的抑制;这可能代表了两种调节剂路径之间的联系。该项目将通过使用三种实验制剂，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，基本上是一种“体内”制剂，允许对细胞内和细胞外环境进行完全的离子和生物化学控制;（ii）鱿鱼神经膜囊泡，一个很好的模型，进行平行运输和生化分析和蛋白质分离;和（iii）鱿鱼轴浆，作为蛋白质分离的来源。具体而言，该研究旨在：（i）确定ATP保护PA调节的质子抑制是否通过ATP调节途径发生，或者是一种独立的ATP效应;（ii）识别和功能表征ATP调节所需的SCRP;（iii）识别和表征与PA刺激相关的结构。关于该项目的广泛影响，必须强调的是，它为青年科学家的培训和教育以及促进美洲和拉丁美洲科学家之间的合作提供了一个极好的机会。

The Sodium/Calcium counter-transport is a ubiquitous structure that reversibly exchanges Sodium for Calcium across cell membranes. This system is conspicuously regulated by intracellular ions and metabolism. Among ions, Sodium and Protons are inhibitory while Calcium stimulates. Regarding metabolism, ATP antagonizes ionic inhibition while, in squid nerve, phosphoarginine (PA) stimulates by promoting the binding of Calcium to its transporting sites. Stimulation by ATP requires a low molecular weight Soluble Cytosolic Regulatory Protein (SCRP); stimulation by PA seems related to another membrane bound protein known as Small Neurofilament. Ionic and ATP regulations take place on a large intracellular region of the exchanger protein. In contrast, PA modulation is likely to occur in other yet unspecified place/s. The experimental evidence indicates that the chemical reactions related to ATP and PA modulations are different. However, the investigators recently found that ATP protects against the inhibition in PA modulation following cellular acidification (increase in Proton concentration); this may represent a link between the two modulators paths. This project will individualize and characterize the interrelationships between ionic and metabolic regulation of the squid Sodium/Calcium exchanger by using three experimental preparations: (i) squid axons under internal dialysis, essentially an "in vivo" preparation that permits complete ionic and biochemical control of intra- and extra cellular environments; (ii) squid nerve membrane vesicles, an excellent model to perform parallel transport and biochemical assays and protein isolation; and (iii) squid axoplasm, as a source for proteins isolation. Specifically the research aims to: (i) establish if ATP protection against proton inhibition of PA modulation takes place via the path for ATP regulation or is an independent ATP effect; (ii) identify and functionally characterize the SCRP required for ATP regulation; and (iii) identify and characterize the structures related to PA stimulation. Regarding the broader impact of the project it must be emphasized that it provides an excellent opportunity for training and education of young scientists and fostering of cooperation between American and Latin American scientists.