COOPERATIVE REGULATION OF THE NA+/H+-ANTIPORTER IN HALOBACTERIUM-HALOBIUM BY DELTA-PH AND DELTA-THETA

The contributions of the transmembrane pH gradient (ΔpH) and electrical potential (Δφ) to the Δ\ ̃gmH+-driven Na+ efflux (mediated by the N,N′-dicyclohexylcarbodiimide-sensitive Na+ H+-antiporter) were investigated in membrane vesicles of Halobacterium halobium. Kinetic analysis in the dark revealed that two different Na+-binding sites are located asymmetrically across the membrane: One, accessible from the external medium, has a Kd (half-maximal stimulation of Na+ efflux) of about <50 mm, and the Na+ binding to the site is a prerequisite for the antiporter activation by Δ\ ̃gmH+. The other cytoplasmic site is the Na+ transport site. The Km for the cytoplasmic Na+ decreased as the ΔpH increased, while the Vmax remained essentially constant in the presence of defined Δφ (140 mV). On the other hand, Δφ elevation above the gating potential (~100 mV) increased the Vmax without changes in the Km in the presence of a fixed ΔpH. It was also noted that the Km value in the absence of Δφ was completely different from and far higher than that observed in the presence of Δφ (>100 mV), indicating the existence of two distinct conformations in the antiporter, resting and Δφ gated; the latter state may be reactive only to ΔpH. On the basis of the present data and the previous data on the pH effect (N. Murakami and T. Konishi, 1989 Arch. Biochem. Biophys. 271, 515-523), a model for the ΔpH-Δφ regulation of the antiporter activation is proposed. © 1990.