Role of (Na,K)-ATPase in osmorregulation and excretion of ammonium ions in euryhaline crustaceans: identification and influence of FXYD2 on the regulation of gill (Na,K)-ATPase of the crab Callinectes danae
This post graduate project is a continuation our line of a long studying program research that has been supported by FAPESP with the aim of the systematic study of the structural properties and kinetics of Na,K-ATPase from gill tissue of different species of crabs and shrimps in order to establish a model for the adaptation of crustaceans to environments of different salinities. The maintained osmolarity of the hemolymph of crustaceans is attributed to the Na,K-ATPase and the mechanism of adaptation suggests that the responses of crustaceans must involve a rapid activation of the transport of ions that must be primary to any other physiological responses of these animals. The current project aims to investigate the cellular signaling linked to the regulation of gill Na,K-ATPase and its direct consequences on the uptake of NaCl during acclimation to hyposaline environments and marine environments. The following studies will be undertaken: 1) correlate the process of acclimation of Callinectes danae with differential expression of FXYD2 (Na,K-ATPase g subunit); 2) study the effect of FXYD2 phosphorylation of Na,K-ATPase of C. danae by protein kinase A; 3) cloning and sequencing of FXYD2 of Na,K-ATPase from C. danae gills; 4) study the effect of polyamines in different saline environments, related to the protein kinases A and C that are activated during osmotic stress, using cultured gill cells from C. danae; 5) elaboration of a protocol for purification of Na,K-ATPase of C. danae in order to obtain an enriched fraction of the enzyme for the identification during proteolytic activation, and subsequently linked to the conformation site binding NH+4; 6) identify the role that other endogenous regulators (dopamine, octopamine) in Na,K-ATPase regulation in cultured gill cells; 7) establish the sequence of aminoacids of Na,K-ATPase in order to identify aminoacid residues involved in cation binding; 8) correlate the FXYD2 with protein kinases stimulated by polyamines and its final action on the activity of Na,K-ATPase in different saline media, using cultured gill cells of C. danae.
News published in Agência FAPESP Newsletter about the scholarship: