Na⁺-H⁺ exchanger of human placental brush-border membrane: Identification and characterization

Syncytiotrophoblast brush-border membrane vesicles isolated from full-term human placentas were shown to transport Na⁺ against a concentration gradient in the presence of an outward proton gradient ([H⁺(i)] > [H⁺](o)). This proton gradient-coupled Na⁺ uptake was markedly inhibited and the uphill transport abolished when the electrochemical proton gradient was dissipated by carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone. The presence of nigericin also eliminated the concentrative uptake of Na⁺ in these vesicles. Dimethylamiloride and harmaline inhibited the proton gradient-induced Na+ uptake. The apparent inhibition constant for this process was 0.32 μM for dimethylamiloride and 100 μM for harmaline. The inhibition by dimethylamiloride was freely reversible and the inhibitor reduced the Na⁺ uptake by directly interacting with the exchange protein rather than by dissipating the H⁺ gradient. The dimethylamiloride-sensitive Na⁺ uptake was saturable with respect to Na⁺. The affinity constant for Na⁺ was 7.8 ± 1.2 mM and the maximal velocity was 38.7 ± 2.4 nmol · mg protein^-1 · min^-1. The dimethylamiloride-insensitive Na⁺ uptake was not saturable and probably represented simple diffusion. The diffusional component accounted for only 10% of the total uptake. Li⁺ strongly competed with Na⁺ for the uptake process and the apparent inhibition constant was 3.6 ± 0.4 mM. Tetraethylammonium also caused significant inhibition of Na⁺ uptake, whereas K⁺, Rb⁺, Cs⁺, and choline had no effect. These data provide evidence for the existence of a Na⁺-H⁺ exchanger in human placental brush-border membrane, and the properties of this exchanger are similar to those of the Na⁺-H⁺ exchanger identified in the brush-border membrane of mammalian kidney and small intestine. The data also show that virtually all of the carrier-mediated Na⁺ uptake observed in placental brush-border membrane vesicles under the experimental conditions occurs via the Na⁺-H⁺ exchange mechanism.