Role in cation translocation of the N-terminus of the alpha-subunit of the Na(+)-K+ pump of Bufo.
1. We have studied the effects on the physiological properties of the Na(+)-K+ pump of both 31- and 40-amino acid N-terminal truncated forms of the alpha-subunit of the Na(+)-K(+)-ATPase. 2. Na(+)-K+ pumps that were moderately ouabain resistant (K1 = 50 microM) were expressed in the Xenopus oocyte by injection of wild-type or truncated variants of the Bufo marinus Na(+)-K(+)-ATPase alpha-subunit cRNA with Bufo beta-subunit cRNA. The function of the Na(+)-K+ pump was studied by electrophysiological methods after Na+ loading and inhibition of the endogenous Xenopus Na(+)-K(+)-ATPase by exposure to a low concentration (0.2 microM) of ouabain. 3. The voltage-dependent potassium activation kinetics of the Na(+)-K+ pump current and the ouabain-sensitive proton-dependent inward current were studied using the two-electrode voltage-clamp technique. A novel technique involving permeabilization of part of the oocyte membrane with digitonin was developed to enable study of the pre-steady-state current following fast voltage perturbation. 4. By comparison with the wild type, the 40-amino acid N-terminal truncation induced a lower level of Na(+)-K+ pump current, a 2- to 3-fold reduction in the apparent external K+ affinity when measured in the presence of extracellular Na+, a relative increase in the proton-dependent inward current, and a reduction in the rate constant of the pre-steady-state current following a voltage step towards a positive membrane potential. The 31-amino acid truncation induced changes that were qualitatively similar but of smaller magnitude. 5. We have analysed these results using a kinetic model of the Na(+)-K+ pump cycle and have shown that all these effects can be explained by the change in a single rate constant in the cycle kinetics, namely a reduction in the rate of the main charge translocating part of the Na(+)-K+ pump cycle, i.e. the forward E1 to E2 conformational change, the deocclusion and release of Na+ to the external side. 6. The highly charged N-terminal segment seems to be directly involved in the mechanism that translocates Na+ ions across the membrane's electrical field.
Wang
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X
Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Jaisser
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F
Horisberger
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002024:Bufo marinus; D002412:Cations / Q000378:metabolism; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D002412:Cations; D004791:Enzyme Inhibitors; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium