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 o f the alpha-subunit of the Na+-K+-ATPase. 2. Na+-K+ pumps that were mo derately ouabain resistant (K-i = 50 mu M) were expressed in the Xenop us 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 me thods after Na+ loading and inhibition of the endogenous Xenopus Na+-K +-ATPase by exposure to a low concentration (0.2 mu M) of ouabain. 3. The voltage-dependent potassium activation kinetics of the Na+-K+ pump current and the ouabain-sensitive proton-dependent inward current wer e studied using the two-electrode voltage-clamp technique. A novel tec hnique involving permeabilization of part of the oocyte membrane with digitonin was developed to enable study of the pre-steady-state curren t 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 externa l K+ affinity when measured in the presence of extracellular Na+, a re lative increase in the proton-dependent inward current, and a reductio n in the rate constant of the pre-steady-state current following a vol tage step towards a positive membrane potential. The 31-amino acid tru ncation 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 ex plained 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 ch ange, 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.