Electrogenic sodium-proton exchange in membrane vesicles from crab (Carcinus maenas) gill

Abstract

Plasma membrane vesicles from Na+-absorbing posterior gills of the green crab Carcinus maenas were shown to contain a Na+-H+ antiporter sensitive to amiloride (Ki = 280 mumol/l), using a fluorometric assay based on acridine orange. Lithium was found to support Li+-H+ exchange, giving Michaelis-Menten kinetics (K0.5 = 20 mmol/l) and a Hill coefficient of 1.32 +/- 0.06, indicating a stoichiometry of 1 Li+:1 H+. The stoichiometry of Na+-H+ exchange, however, appeared to be 2 Na+:1 H+. The dependence of exchange rate on Na+ concentration followed a sigmoid relationship (K0.5 = 34 mmol/l), producing a Hill coefficient of 1.99 +/- 0.18. Measurements made with the potential-sensitive dye 3,3'-dipropylthiadicarboxocyanine iodide directly demonstrated electrogenicity of Na+-H+ exchange. Na+-loaded vesicles showed greater potential change upon addition to Na+-free medium than upon addition to Na+-containing medium, and the developed potential was reversed by the addition of external Na+. Amiloride reduced the rate of membrane polarization and abolished the reversal by external Na+. The crustacean Na+-H+ antiporter therefore bears similarities to the vertebrate antiporter but is uniquely electrogenic.