The effect of membrane potential on the mammalian sodium‐potassium pump reconstituted into phospholipid vesicles.

1. 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)