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Charge Translocation by the Na⁺/K⁺ Pump under Na⁺/Na⁺ Exchange Conditions: Intracellular Na⁺ Dependence

Miguel Holmgren ^* and Robert F. Rakowski 

^* National Institutes of Health, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland; and Department of Biological Sciences, Ohio University, Athens, Ohio

Correspondence: Address reprint requests to R. F. Rakowski, Tel.: 740-593-2330; Fax: 740-593-0300; E-mail: rakowski{at}ohio.edu.

The effect of intracellular (i) and extracellular (o) Na⁺ on pre-steady-state transient current associated with Na⁺/Na⁺ exchange by the Na⁺/K⁺ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from –180 to +100 mV in the absence of external Na⁺ were subtracted from current records obtained under Na⁺/Na⁺ exchange conditions. Na⁺-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (k_tot) of the slow component of the -sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na⁺ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na⁺ was halved, the midpoint voltage of the charge distribution (V_q) shifted –25.3 ± 0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance = 0.69 ± 0.01. The effect of changes of on -sensitive transient current was investigated. The midpoint voltage (V_q) of the charge distribution curve was not affected over the concentration range 3.13–50 mM. As was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent K_m of 3.2 ± 0.2 mM. The effects of lowering on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na⁺ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na⁺ binding and extracellular Na⁺ release.