Ionic dependence of active Na-K transport: 'Clamping' of cellular Na⁺ with monensin

The Na⁺ ionophore monensin was used to study the Na⁺- and K⁺-dependence of ouabain-inhibitable ⁸⁶Rb⁺ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na⁺ that were stable for up to 2 h. In experiments in which cellular Na⁺ was thus 'clamped' at various levels, the activation curve for ouabain-inhibitable ⁸⁶Rb⁺ uptake as a function of intracellular Na⁺ was found to be steepest near basal Na⁺ levels (Hill coeficient ≃ 2.4), indicating that these cells can respond to relatively large changes in passive Na⁺ entry by increasing the rate of Na-K pump function with only minimal increases in cellular Na⁺. Exposure of cells to monensin also permitted examination of the extracellular-K⁺ dependence of ouabain-inhibitable ⁸⁶Rb⁺ uptake in the presence of saturating intracellular Na⁺ and yielded a Hill coefficient of ~1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable ⁸⁶Rb⁺ uptake in intact cells was similar to the enzymatic V(max) of the Na⁺-K⁺-ATPase in cell lysates, suggesting that the Na⁺-K⁺-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.