Cooperative regulation of the Na+/H(+)-antiporter in Halobacterium halobium by delta pH and delta phi.
The contributions of the transmembrane pH gradient (delta pH) and electrical potential (delta phi) to the delta mu H(+)-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 less than 50 mM, and the Na+ binding to the site is a prerequisite for the antiporter activation by delta mu H+. The other cytoplasmic site is the Na+ transport site. The Km for the cytoplasmic Na+ decreased as the delta pH increased, while the Vmax remained essentially constant in the presence of defined delta phi (140 mV). On the other hand, delta phi elevation above the gating potential (approximately 100 mV) increased the Vmax without changes in the Km in the presence of a fixed delta pH. It was also noted that the Km value in the absence of delta phi was completely different from and far higher than that observed in the presence of delta phi (greater than 100 mV), indicating the existence of two distinct conformations in the antiporter, resting and delta phi gated; the latter state may be reactive only to delta pH. 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 delta pH-delta phi regulation of the antiporter activation is proposed.
Murakami
N
N
Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Japan.
Konishi
T
T
D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006217:Halobacterium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium