Pre-steady-state transient currents mediated by the Na/K pump in internally perfused Xenopus oocytes.
Pre-steady-state transient currents have been investigated in the vegetal pole of Xenopus oocytes using the open-oocyte vaseline-gap technique of Taglialatela, Toro, and Stefani (Biophysical Journal. 61:78-82, 1992). Voltage pulses 40 ms in duration were made from a holding potential of -40 mV to command potentials over the range -160 to +60 mV in increments of 20 mV. Current records (averaged 20X; sampled every 200 microseconds) in the presence of dihydroouabain (DHO) or absence of external Na+ (Nao) were subtracted from current records obtained under Na/Na exchange conditions, i.e. internally perfused with 50 mM Na+, 5 mM ATP, and 5 mM ADP (K(+)-free) and externally superfused with 100 mM Na+,K(+)-free solution. Transient currents were dependent on intracellular Na+ and nucleotides, and diminished by activation of forward pumping; they were also reduced by 10 micrograms ml-1 of oligomycin B applied to the external solution. These properties of the pre-steady state currents are consistent with the Na/K pump operating in its electroneutral Na/Na exchange mode. The voltage dependence of the DHO- and Nao-sensitive transient currents was analyzed using a pseudo two-state model in which only the rate coefficient for Nao-binding/reocclusion is voltage-dependent (Rakowski, R. F. 1993. J. Gen. Physiol. 101:117-144). The apparent valence of the charge moved during the on (zq-on) and off (zq-off) of the pulse were 0.96 +/- 0.05 and 0.95 +/- 0.05 for Nao-sensitive, and 1.10 +/- 0.07 and 0.85 +/- 0.06 for DHO-sensitive transient currents, respectively. The total amount of charge moved (Qtot) and the mid-point voltage of the charge distribution (Vq) were 230 +/- 15 pC and -56.2 +/- 5.1 mV, and 268 +/- 34 pC and -67.0 +/- 7.6 mV for Nao- and DHO-sensitive transient currents, respectively. The apparent valence (zk) and the voltage at which the forward and backward rates are equal (Vk) obtained from the relaxation rates were 0.80 +/- 0.05 and -129.3 +/- 10.0 mV, and 0.86 +/- 0.10 and -135.1 +/- 9.0 mV for the Nao- and DHO-sensitive pre-steady state currents, respectively. The values of the parameters were not statistically significantly different between the Nao- and DHO-sensitive transient currents. Excluding the first 600 microseconds after the onset of a voltage step which was not temporally resolved, transient currents showed no indication of a rising phase. These results support the idea that charge translocation occurs within an external access channel at a rate that is governed by a voltage-dependent binding/reocclusion process and a voltage-independent deocclusion/unbinding process.
Department of Physiology and Biophysics, Chicago Medical School, North Chicago, Illinois 60064.
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Holmgren
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Rakowski
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D008954:Models, Biological; D009840:Oligomycins / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000031:analogs & derivatives / Q000494:pharmacology; D010477:Perfusion; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D014982:Xenopus laevis
D009840:Oligomycins; C000977:dihydroouabain; C048335:oligomycin B; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium