Regulation of endogenous and expressed Na⁺/K⁺ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases

Summary

Modulation of the current generated by the Na⁺/K⁺ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I–V) relationship of the Na⁺/K⁺ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent K _m value for pump stimulation by external [K⁺] (Rakowski et al., 1991). To study this feature in more detail. Xenopus oocytes were used for comparative analysis of the negative slope of the I–V relationship of the endogenous Na⁺/K⁺ pump and of the Na⁺/K⁺ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na⁺-free solution and in the presence of high concentrations of Ba²⁺ and tetraethylammonium, to block all nonpump related K⁺-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K⁺.

The voltage dependence of the K _m value for stimulation of the Na⁺/K⁺ pump by external [K⁺] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the K _m value for the Na⁺/K⁺ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K⁺] and can be interpreted by voltage-dependent K⁺ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge.

Application of 1,2-dioctanoyl-sn-glycerol (diC₈. 10–50 μm). which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K⁺ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent K _m at 0 mV increased from 0.46 to 0.83 mm. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge.

Injection of cAMP (final concentration 50 μm), which stimulates protein kinase A, has an effect opposite to stimulation of protein kinase C. The activity of the Xenopus Na⁺/K⁺ pump is elevated by 80%, and the voltage dependence of K⁺ stimulation becomes less pronounced. For the endogenous pump the apparent effective charge decreased from 0.56 to 0.38 of elementary charge and the apparent K _m at 0 mV decreased from 0.78 to 0.65 mm. Also for the Torpedo pump, the effective charges and apparent affinities became reduced.

The data suggest that species differences in voltage-dependent stimulation of the Na⁺/K⁺ pump by external K⁺ can account for differences in the steepness of the negative slope in the I–V relationships observed in different preparations. In addition, they suggest that the voltage dependence and the maximum activity of the Na⁺/K⁺ pump can be modulated by activation of protein kinases.