Kinetic mechanism of ATP action in Na(+)-K(+)-Cl- cotransport of HeLa cells determined by Rb+ influx studies

Abstract

The kinetics of Na(+)-K(+)-Cl- cotransport were studied by measuring ouabain-insensitive furosemide-sensitive Rb+ influx (JRb) into HeLa cells while varying the cellular ATP and the extracellular Rb+ and Na+ concentrations. Results reveal that ATP stimulates JRb by increasing the affinity of the cotransporter for Rb+ (K+), and the apparent Michaelis constant (Km) for ATP was 0.95 +/- 0.03 mmol/l cell water. Two ATP molecules may relate to the uptake of one Rb+ by the cotransport pathway, as examined by the nonlinear least-squares method for goodness-of-fit and a Hill plot, JRb was strengthened by an increase in the inward chemical gradient associated with cell swelling on preincubation in a low-Na+ high-K+ medium, accompanying an increase in the affinity of the transporter for ATP. JRb was apparently activated by extracellular Na+, and the activation was enhanced by an increase in the cellular ATP concentration. Lactate production stimulated by 2 microM carbonylcyanide m-chlorophenyl hydrazone (CCCP) was reduced by 10 microM ouabain but not altered by further addition of 0.1 mM furosemide. Increases in cellular adenosine 3',5'-cyclic monophosphate (cAMP) caused by treatment with 0.1 mM isoproterenol plus 0.5 mM 3-isobutyl-1-methylxanthine or with 0.1 mM dibutyryl cAMP did not influence JRb. From this and previous studies, we propose a general and a specific model of Na(+)-K(+)-Cl- cotransport, which elucidate the order of binding of extracellular ions and reaction of cellular ATP.