Kinetic parameters and mechanism of active cation transport in HeLa cells as studied by Rb+ influx

On incubation of HeLa cells in chilled isotonic medium, intracellular Na⁺ (Na_c⁺) increased and K⁺ (K_c⁺) decreased with time, reaching steady levels after 3 h. The steady levels varied in parallel with the extracellular cation concentrations (Na⁺]_e, K⁺]_e). The cell volumes and the protein and water contents, respectively, of cells kept for 3 h in chilled media of various Na⁺]_e and K⁺]_e were not significantly different. Ouabain-sensitive Rb⁺ influx took place at the initial rate for a certain period which depended on Na⁺]_c at the beginning of the assays. The existence of two external K⁺ loading sites per Na⁺/K⁺-pump was demonstrated. The affinities of the sites for Rb⁺ as a congener of K⁺ were almost the same. Na_e⁺ inhibited ouabain-sensitive Rb⁺ influx competitively, whereas K_c⁺ was not inhibitory. Kinetic parameters were determined: the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for Rb_e⁺ in the absence of Na_e⁺ was 0.16 mM and the K_i for Na_e⁺ was 36.8 mM; the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 19.5 mM and the K_i for K_c⁺ seemed to be extremely large. The rate equation of the ouabain-sensitive Rb⁺ influx suggests that Na⁺ and K⁺ are exchanged alternately through the pump by a binary mechanism.