Asymmetry of the Na+/H+ antiport of dog red cell ghosts. Sidedness of inhibition by amiloride.

Abstract

Amiloride is a potent inhibitor of the Na+/H+ antiport. Inhibition is generally competitive with extracellular Na+ and therefore believed to result from binding to the outward-facing transport site. It is not known whether amiloride can interact with the internal aspect of the antiport. This question was addressed by trapping the drug inside resealed dog red cell ghosts. The antiport, which is quiescent in resting ghosts, was activated by acid-loading the cytoplasm. This was accomplished by exchanging extracellular Cl- for internal HCO-3 through capnophorin, the endogenous anion exchanger. The activity of the Na+/H+ antiport was detected as an increase in cell volume, resulting from the net osmotic gain associated with coupled Na+/H+ and Cl-/HCO-3 exchange, or as the uptake of 22Na+. Intracellular amiloride, at concentrations in excess of 100 microM, failed to inhibit Na+/H+ exchange. This is approximately 10 times higher than the concentration required for half-maximal inhibition when amiloride is added externally. Independent experiments demonstrated that failure of internal amiloride to inhibit exchange was not due to leakage of the inhibitor, to differences in pH, or to binding or inactivation of amiloride by the soluble contents. It was concluded that the antiport is functionally asymmetric with respect to amiloride. This implies that the transport site undergoes a conformational change upon translocation across the membrane or, alternatively, that a second site required for amiloride binding is only accessible from the outside.