Kinetic study on the effects of intracellular K+ and Na+ on Na+, K+, Cl- cotransport of HeLa cells by Rb+ influx determination.

The effects of intracellular K+ and Na+ (K+c, Na+c) on the Na+, K+, Cl- cotransport pathway of HeLa cells were studied by measuring ouabain-insensitive, furosemide-sensitive Rb+ influx (JRb) at various intracellular concentrations of K+ and Na+ ([K+]c, [Na+]c). When [K+]c was increased and [Na+]c was decreased, keeping the sums of their concentrations almost constant, JRb as a function of the extracellular Rb+ or Na+ concentration ([Rb+]e, [Na+]e) was stimulated. However, the apparent K0.5 for Rb+e or Na+e remained unchanged and the ratio of the apparent K0.5 for K+c and the apparent Ki for Na+c was larger than 1. When JRb was increased by hypertonicity by addition of 200 mM mannitol, the apparent maximum JRb increased without change in the apparent K0.5 for Rb+e. These results show that K+c stimulates and Na+c inhibits JRb, without change in the affinities of the pathway for Rb+e and Na+e. The affinity for K+c is slightly lower than that for Na+c. Hypertonicity enhances JRb without any change in the affinity for Rb+e. We derived a kinetic equation for JRb with respect to K+c and Na+c and proposed a general and a special model of the pathway. The special model suggests that, in HeLa cells, JRb takes place when Rb+e binds to the external K+ binding site of the pathway after the binding of K+c to the internal regulatory site.