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

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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.