We have studied effects of electrical diffusion potentials on active Na+-K+ exchange in phospholipid vesicles reconstituted with pig kidney Na+, K+-ATPase. 2. Diffusion potentials, negative inside, were established using outwardly directed K+ gradients plus valinomycin or Li+ gradients plus a Li+ ionophore, AS701. Measurement of fluorescence changes of the carbocyanine dye DiS-C3-(5) showed that the ionophores generated potentials of the expected orientation and of sufficient stability for their effects on active transport to be assessed. Measurement of rates of passive 22Na+ fluxes, over a wide range of diffusion potentials, were consistent with the quantitative predictions of the constant-field flux equation. This result demonstrates that values of diffusion potentials calculated from the Nernst or constant-field equation are accurate. 3. In some conditions, the inside-negative potential (-130 to -180 mV) accelerated the rate of ATP-dependent Na+-K+ exchange on inside-out-oriented pumps, compared to 'control' without the ionophores. Reduction in the size of the diffusion potentials by addition to the medium of Li+ with AS701 or Cs+ with the valinomycin progressively annulled the acceleratory effects, consistent with these being true effects of a change in membrane potentials. 4. At saturating cytoplasmic Na+ and ATP concentrations, the diffusion potential accelerated ATP-dependent Na+-K+ exchange by up to about 30% compared to control but this effect disappeared at rate-limiting ATP concentrations (approximately 1 microM). 5. Using prior knowledge of rate-limiting steps, we interpret this finding to mean that the conformational transition E2(2K)----E12K associated with transport of two K+ ions is voltage insensitive while E1P(3Na)----E2P3Na associated with transport of three Na+ ions is voltage sensitive. The simplest explanation is that the net charge in the transport domain of the protein when no ions, 2K+ or 3Na+ are bound is -2, 0 and +1 respectively. 6. The accelerating effect of the negative-inside diffusion potential on Na+-K+ exchange is greater at limitingly low cytoplasmic Na+ concentrations than at saturating cytoplasmic Na+ concentrations. Cytoplasmic Na+ activation curves show that the diffusion potential increases the apparent cytoplasmic Na+ affinity and reduces the sigmoidicity of cytoplasmic Na+ activation. 7. A kinetic analysis reveals that this effect on apparent affinity is due to an increase in intrinsic Na+ binding and occurs in addition to the effect on a transport rate constant.(ABSTRACT TRUNCATED AT 400 WORDS)
