To investigate the voltage dependence of the Na+/K+ pump, current-voltage relations were determined in prophase-arrested oocytes of Xenopus laevis. All solutions contained 5 mM Ba2+ and 20 mM tetraethylammonium (TEA) to block K+ channels. If, in addition, the Na+/K+ pump is blocked by ouabain, K(+)-sensitive currents no larger than 50 nA/cm2 remain. Reductions in steady-state current (on the order of 700 nA/cm2) produced by 50 microM ouabain or dihydro-ouabain or by K+ removal, therefore, primarily represent current generated by the Na+/K+ pump. In Na(+)-free solution containing 5 mM K+, Na+/K+ pump current is relatively voltage independent over the potential range from -160 to +40 mV. If external [K+] is reduced below 0.5 mM, negative slopes are observed over this entire voltage range. Similar results are seen in Na(+)- and Ca(2+)-free solutions in the presence of 2 mM Ni2+, an experimental condition designed to prevent Na+/Ca2+ exchange. The occurrence of a negative slope can be explained by the voltage dependence of the apparent affinity for activation of the Na+/K+ pump by external K+, consistent with the existence of an external ion well for K+ binding. In 90 mM Na+, 5 mM K+ solution, Na+/K+ pump current-voltage curves at negative membrane potentials have a positive slope and can be described by a monotonically increasing sigmoidal function. At an extracellular [K+] of 1.3 mM, a negative slope was observed at positive potentials. These findings suggest that in addition to a voltage-dependent step associated with Na+ translocation, a second voltage-dependent step that is dependent on external [K+], possibly external K+ binding, participates in the overall reaction mechanism of the Na+/K+ pump.

Full-grown prophase-arrested oocytes of Xenopus laevis were treated with 50 nM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or with 50 nM 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) that does not activate protein kinase C. The effect on membrane currents and capacitance, inulin uptake and ouabain binding, and on membrane morphology were analyzed. (i) During application of PMA, current generated by the Na+/K+ pump decreases; in addition, Cl- and K+ channels become inhibited. This general decrease in membrane conductance reaches steady state after about 60 min. 4 alpha PDD was ineffective. (ii) Ouabain binding experiments demonstrate that PMA (K1/2 = 7 nM), but not 4 alpha PPD, induces a reduction of the number of pump molecules in the surface membrane. Permeabilization of oocytes by digitonin plus 0.02% SDS renders all binding sites present prior to PMA treatment again accessible for ouabain. The KD value for ouabain binding is not influenced. 4 alpha PDD was ineffective. (iii) Exposure of oocytes to PMA reduces membrane capacitance and stimulates uptake of inulin suggesting an increase in endocytosis. Electron micrographs show that PMA reduces the number and length of microvilli, leading finally to a smooth membrane surface with a reduced surface area. From these results we conclude that stimulation of protein kinase C leads to downregulation of the sodium pump. A major portion of this inhibition is brought about by reduction in area of surface membrane with a concomitant internalization of pump molecules. In addition to this mode of downregulation, a direct effect of stimulation of protein kinase C on the pump molecule cannot be excluded.

No abstract

The serum and glucocorticoid-dependent kinase-1 (sgk1) is expressed in a wide variety of tissues including renal epithelial cells. As it is up-regulated by aldosterone, it is considered to participate in the regulation of renal Na(+) reabsorption. Indeed, co-expression of sgk1 with the renal epithelial Na(+) channel (ENaC) augments Na(+) channel activity. The aim of the present study was to examine possible effects of sgk1 on Na(+)/K(+)-ATPase activity. To this end dual-electrode voltage-clamp experiments were performed in Xenopus oocytes expressing the active kinase (S422D)sgk1 or the inactive mutant (K127N)sgk1. Na(+)/K(+)-ATPase activity was estimated from the hyperpolarization (delta V(m)) and the outwardly-directed current ( I(P)) created by addition of extracellular K(+) in the presence of K(+) channel blocker Ba(2+). Both delta V(m) and I(P) were significantly larger in oocytes expressing (S422D)sgk1 than in those expressing (K127N)sgk1 or having been injected with water. I(P) was fully inhibited by ouabain. Ion-selective microelectrodes showed that the stimulation of pump current was not the result of altered cytosolic Na(+) activity or pH. The present results thus point to an additional action of sgk1 that may participate in the regulation of renal tubular Na(+) transport. Moreover, sgk1 may be involved in the regulation of Na(+)/K(+)-ATPase in extrarenal tissues.

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 Km 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 Km 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 Km 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 microM), 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 Km 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.(ABSTRACT TRUNCATED AT 400 WORDS)