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

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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.