title: Proton dependence of the partial reactions of the sodium-proton exchanger in renal brush border membranes.
abstract: The pre-steady state time dependence of Na+ accumulation by the Na(+)-H+ exchanger in renal brush border membrane vesicles was investigated at 0 degree C by a manual mixing technique using amiloride to quench the reaction. Dilution of acid-loaded (pHi 5.7) vesicles into an alkaline medium (pHo 7.7) containing 1 mM 22Na+ produced a time course of amiloride-sensitive Na+ uptake that consisted of three distinct phases: 1) a lag, 2) a monoexponential "burst," and 3) a linear or steady state phase. Experiments testing for the presence of 22Na+ backflux, residual Na+ binding to the membrane, and hysteresis were negative, lending support to the hypothesis that the burst phase corresponds to Na+ translocation during the initial turnover of Na(+)-H+ exchanger. Lowering the internal pH increased the amount of na+ uptake in each of the phases without affecting the apparent burst rate, whereas lowering the external pH inhibited Na+ uptake while increasing the duration of the lag phase. The pattern of inhibition produced by external H+ was of the simple competitive type, indicating that Na+ and H+ share a common binding site. Steady state Na+ uptake showed a sigmoidal dependence on internal pH (Hill coefficient = 1.67), consistent with the presence of an internal allosteric H+ activation site. Alkaline loading conditions (pHi 7.7), which favor desaturation of the internal H+ binding sites, completely abolished Na+ uptake in the steady state. In contrast, Na+ accumulation during the burst phase was reduced to 25% of an acid-loaded (pHi 5.7) control. The persistence of the burst phase and the disappearance of steady state Na+ uptake under alkaline loading conditions suggest that recycling of the H(+)-loaded exchanger is a late event in the transport cycle that follows Na+ translocation (ping-pong mechanism) and controls the steady state rate of Na+ accumulation. Activation of the recycling step involves sequential binding of H+ to the allosteric and transport sites, thus accounting for the cooperative dependence of steady state Na+ uptake on the internal [H+].
authors.affiliation: Charles H. Best Institute, Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Otsu
authors.forename: J L
authors.initials: JL
authors.lastname: Kinsella
authors.forename: E
authors.initials: E
authors.lastname: Koh
authors.forename: J P
authors.initials: JP
authors.lastname: Froehlich
mesh_terms: D000495:Allosteric Site; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D006096:Gramicidin; D000584:Amiloride; D012964:Sodium