Mechanism of function of dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter in Halobacterium halobium: pH effect.
The regulatory roles of medium pH, a transmembrane pH gradient (delta pH), and an electrical potential (delta phi) on the activation of the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter were studied in the membrane vesicle of Halobacterium halobium in the dark. Neither delta pH nor delta phi independently activated the antiporter but a combination could. The initial rate of Na+ extrusion did not proportionally relate to the size of delta microH+ imposed. The delta microH+-coupled Na+ efflux in the presence of delta phi (-140 mV) increased as external pH decreased, regardless of the size of delta pH, suggesting the existence of one external H+-binding site (apparent pKa 4.6) whose protonation determines primarily the Na+/H+-exchange activity. On the other hand, the dependence of the Na+ efflux on cytoplasmic pH varied with the size of delta pH imposed and the apparent pKa for the cytoplasmic H+ increased with elevating delta pH. The resulting pKa difference across the membrane seems to be the key mechanism for the facilitation of Na+-coupled H+ influx. In other words, delta pH modulates Na+/H+-exchange activity through manipulating the H+ affinity on the cytoplasmic regulatory site. The Na+ extrusion was gated by the threshold delta phi of -100 mV regardless of the size of existing delta pH. delta phi acts on the protonated antiporter and converts it into an active state which becomes delta pH reactive.
Murakami
N
N
Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Japan.
Konishi
T
T
D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004563:Electrochemistry; D006217:Halobacterium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017923:Sodium-Hydrogen Exchangers
D002234:Carbodiimides; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D004024:Dicyclohexylcarbodiimide