Essential activation of Na(+)-H+ exchange by [H+]i in HL-60 cells

Abstract

The intracellular pH (pHi) dependence of the rate of Na(+)-H+ exchange was determined in undifferentiated promyelocytic HL-60 cells by measuring alkalinization rates using the fluorescent pHi indicator 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). BCECF was calibrated in the pH range from 5 to 7 using the nigericin technique of Thomas and co-workers (J. A. Thomas, R. N. Buchsbaum, A. Zimniak, and E. Racker. Biochemistry 18: 2210-2218, 1979). Exchange rate increases as pHi is lowered below pH 7.00. At low pH (pH below 6.3), the dependence of Na(+)-H+ exchange rate on intracellular proton activity is well fitted by the Michaelis-Menten equation with a maximum exchange velocity of 33.7 +/- 2.4 mmol H(+).1 cell water-1.min-1 and a half-saturation constant of 1.35 +/- 0.28 microM (corresponding to a minus log of the Michaelis constant of 5.89). However, a Hill plot reveals that the Hill coefficient changes gradually from one to two when pH is changed from 5 to 7, ruling out Michaelian kinetics. The dependence of exchange flux on internal protons is well fit in the full pH range from 5 to 7 by a simple kinetic model (essential activation) with modifier and transport sites for internal proton binding. At low pH, failure to correct BCECF measurement of pHi for contribution to fluorescence signal from extracellular dye and for quenching of intracellular BCECF leads to an artifactual increase in the measured Hill coefficient. These two findings (increase in Hill coefficient as pHi is increased and artifactual increase in Hill coefficient because of methodological reasons) provide a good explanation for the wide range of Hill coefficients reported in the literature.