Physiological characterization of the Na⁺/Ca²⁺ exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

The purpose of this study was to physiologically characterize the basolateral Na⁺/Ca²⁺ exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na⁺-dependent Ca²⁺ uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na⁺-dependent Ca²⁺ uptake rate into BLMV was temperature insensitive. Na⁺-dependent Ca²⁺ uptake rate was dependent upon free Ca²⁺ with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J_max) = 2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K_m) = 0.69 μM Ca²⁺; antennal gland, J_max = 13.2 nmol/mg per min, K_m = 0.59 μM Ca²⁺. The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na⁺-dependent Ca²⁺ uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 μM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 μM in hepatopancreas and was stimulatory at 1000 μM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na⁺-dependent Ca²⁺ uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca²⁺ ATPase (PMCA), the NCX has 70-fold greater J_max (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J_max and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca²⁺ efflux in intermolt.