Sodium dependence of the Na(+)-H+ exchanger in the pre-steady state. Implications for the exchange mechanism

The pre-steady state time course of amiloride-sensitive Na+o uptake by the Na(+)-H+ exchanger in renal brush border membrane vesicles (BBMV) exhibits a burst phase at 0 degrees C which corresponds to the initial turnover of the exchanger (Otsu, K., Kinsella, J. L., Sacktor, B. S., and Froehlich, J. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4818-4822). Investigation of the Na+o dependence of the Na(+)-H+ exchanger between 1 and 10 mM Na+ revealed that activation of the burst phase involves at least two Na+ transport sites interacting with positive cooperativity. In this study, characterization of the Na+ transport sites contributing to the burst phase was extended to include Na+ concentrations below 1 mM. Between 0.1 and 1 mM Na+ the amplitude of the burst phase in acid-loaded BBMV (pHi 5.7; pHo 7.7) exhibited a sigmoidal dependence on [Na+]o, consistent with the presence of a second class of high affinity Na+ transport sites with cooperative binding characteristics. In contrast, steady state Na+ uptake obeyed Michaelis-Menten kinetics, similar to the behavior observed previously at higher (1-10 mM) Na+o concentrations. Treatment of the vesicles with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, which induced the formation of an inside-negative membrane potential, increased the burst amplitude but had no effect on the steady state uptake velocity. Experiments performed with alkaline-loaded BBMV (pHi 7.7; pHo 7.7), which permit only a single turnover of the exchanger, gave a simple hyperbolic dependence of the burst amplitude on [Na+]o (0.5-5 mM). We propose that the change in multiplicity of Na+ transport sites and membrane potential sensitivity that occurs in the transition between the pre-steady state and the steady state of Na+ uptake in acid-loaded vesicles reflects the presence of an oligomer which operates according to a "flip-flop" mechanism. The minimum subunit composition inferred from the biphasic [Na+]o dependence of the burst amplitude is a dimer at low (< 1 mM) Na+o levels and a tetramer at high [Na+]o. Communication between the subunits producing the complex [Na+]o dependence is controlled by the intravesicular (cytoplasmic) H+ modifier site. Under alkaline conditions (pH 7.7), where this site is unoccupied, the subunits behave as independent units and cease operation after the first turnover. Occupation of the H+ modifier site activates a conformational interaction between the subunits that leads to cooperative Na+o binding, alternation of the transport sites, and repetitive cycling of the Na(+)-H+ exchanger.