SODIUM AND CALCIUM SHARE THE ELECTROGENIC 2 NA+-1 H+ ANTIPORTER IN CRUSTACEAN ANTENNAL GLANDS

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient ([H]i > [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pH(i) = 5.0 and pH(o) = 8.0 and followed the Hill equation for binding cooperativity [apparent maximal influx (J(max)) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (K(Na)) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (K(i1) = 14-mu-M; K(i2) = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (K(i1) = 20 mu-M; K(i2) = 500-mu-M). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (K(i1) = 70-mu-M; K(i2) = 500-mu-M) and Na (K(i1) = 12 mM; K(i2) = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.