Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes.
This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials. ATP-dependent Nacyt/Kexc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent 22Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Nacyt (2-50 mM) and ATP (1-1000 microM) and pH's (6.5-8.5), the classical 3Nacyt/2Kexc coupling ratio is maintained. However, at low Nacyt concentrations (less than 0.8 mM), the coupling ratio is apparently less than 3Nacyt/2Kexc. ATP-dependent Nacyt/congenerexc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Naexc, act as Kexc surrogates in an electrogenic 3Nacyt/2congenerexc exchange. (ATP + Pi)-dependent Kcyt/Kexc exchange in K-loaded vesicles is electroneutral. ATP-dependent "uncoupled" Na flux into Na- and K-free vesicles is electroneutral at pH 6.5-7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The 22Na flux shows no anion specificity. We propose that "uncoupled" Na flux is an electroneutral 3Nacyt/3Hexc exchange at pH 6.5-7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve Kcyt/Hexc exchange. We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Nacyt in Na/K exchange and Hexc in "uncoupled" Na flux, coupling ratios may change.
Goldshleger
R
R
Biochemistry Department, Weizmann Institute of Science, Rehovoth, Israel.
Shahak
Y
Y
Karlish
S J
SJ
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D001693:Biological Transport, Active; D004396:Coloring Agents / Q000378:metabolism; D007555:Isoxazoles / Q000378:metabolism; D007679:Kidney Medulla / Q000201:enzymology*; D008564:Membrane Potentials; D008954:Models, Biological; D011188:Potassium / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine
D004396:Coloring Agents; D007555:Isoxazoles; D011510:Proteolipids; C020485:proteoliposomes; C031184:oxonol VI; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium