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Abstract

To investigate the voltage dependence of the Na⁻/K⁻ pump, current-voltage relations were determined in prophasearrested oocytes ofXenopus laevis. All solutions contained 5mm Ba²⁻ and 20mm 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 μm ouabain or dihydro-ouabain or by K⁺ removal, therefore, primarily represent current generated by the Na⁻/K⁻ pump. In Na⁻-free solution containing 5mm K⁺, Na⁺/K⁺ pump current is relatively voltage independent over the potential range from −160 to +40 mV. If external [K⁺] is reduced below 0.5mm, negative slopes are observed over this entire voltage range. Similar results are seen in Na⁺- and Ca²⁺-free solutions in the presence of 2mm 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 90mm Na⁺, 5mm 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 1.3mm, 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.