Stimulating Effect of an Artificial Electron Mediator, Phenazine Methosulfate, on Rb⁺ Transport in HeLa Cells

Abstract

To obtain evidence of the action of the intracellular redox system as a regulating factor of membrane K⁺ transport in HeLa cells, we used an artificial electron mediator, phenazine methosulfate (PMS). An addition of PMS to the incubation medium increased the intracellular accumulation of Rb⁺ as an analog of K⁺. This increase, which was ouabainsensitive, was shown to be due to the stimulation of active Rb+ transport. The rate of ouabainsensitive Rb⁺ uptake was a linear function of the cellular bulk ATP level. When cells were treated with 20 μM PMS, the linear curve showed a parallel shift upward. The range of the shift, the PMS-stimulated part of the Rb⁺ uptake, was independent of the ATP level. The intracellular level of Rb⁺ plus K⁺, which was equal to the K⁺ level in cells incubated in normal medium, was elevated slightly, but Na⁺ level remained unchanged. These results indicated that the stimulation of Rb⁺ uptake had no effect on changes in the intracellular levels of monovalent cations. The maximal rate of ouabainsensitive Rb⁺ uptake, Jmax, was increased, but the apparent Km in relation to extracellular Rb+ was unaffected. The specific binding of [³H]ouabain was not enhanced. Thus, the total number of Rb⁺ binding sites and the affinity of Rb⁺ to those sites would not be affected significantly. We concluded that the stimulating action of PMS is due either to the acceleration of Rb+ pumping at each Na⁺, K⁺-pump or to activation of the resting pumps.