Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles.
A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.
Clarke
R J
RJ
Department of Biology, University of Konstanz, F.R.G.
Apell
H J
HJ
Läuger
P
P
D000818:Animals; D001693:Biological Transport, Active; D066298:In Vitro Techniques; D007555:Isoxazoles; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids; D008564:Membrane Potentials; D011188:Potassium / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D007555:Isoxazoles; D008081:Liposomes; D008563:Membrane Lipids; C031184:oxonol VI; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium