PARIKH, P.C.**; TIERNEY, K.J.; GERBER, N.L.; LOVETT, D.L.; The College of New Jersey, Ewing: Na+,K+-ATPase activity in gills of the green crab Carcinus maenas may be modulated by membrane trafficking during salinity change
The activity of Na+,K+-ATPase (ATPase) increases substantially in estuarine crabs acclimated to dilute seawater. Although crabs hyperosmoregulate soon after transfer to low-salinity water, no change in ATPase activity in gill homogenates typically is detected during acute exposure. We hypothesize that the amount of ATPase enzyme within the plasma membrane is increased during acute exposure of crabs to low-salinity seawater by fusing vesicles (which had been sequestered within the cytoplasm while the crab was in higher-salinity seawater) with the plasma membrane. To test whether membrane trafficking was occurring, we perfused contralateral gills of green crabs Carcinus maenas acclimated to 10 ppt salinity with either 10 or 32 ppt saline while immersing them in seawater of the corresponding salinity. After 1 hour, gills were perfused with saline to which biotin had been introduced, and then gills were flushed with saline alone to remove any unbound biotin. In theory, biotin would have bound to portions of the plasma membrane in contact with the hemocoel, but it would not have bound to any membrane that had been sequestered within the cytoplasm. Biotinylated membrane was isolated from homogenates of each gill with paramagnetic beads conjugated with streptavidin. Preliminary tests have demonstrated that the streptavidin treatment removed a greater proportion of the ATPase activity from homogenates of low-salinity gills than from high-salinity gills. Therefore, ATPase appears to be trafficked into the cytoplasm in high-salinity conditions, where it would be unavailable for transporting ions across the plasma membrane.