Sidedness of the effects of sodium and potassium ions on the conformational state of the sodium—potassium pump

Abstract

(Na,K)ATPase from [pig] kidney membranes was reconstituted into proteoliposomes following solubilization in cholate, by the freeze-thaw sonication procedure described by Kasahara et Hinkle (1977). The method is rapid and convenient. Upon addition of ATP to the exterior medium the reconstituted vesicles sustain high rates of active 22Na uptake and 86Rb efflux with many properties similar to those of the Na/K pump in well characterized cells such as erythrocytes. Observations on both active and passive transport of 22Na and 86Rb indicate that the vesicle population is heterogeneous; about 40% contain Na/K pumps and the remainder, plain lipid vesicles. The major Na+- or K+-stabilized non-phosphorylated conformational forms of the (Na,K)ATPase, E1 .cntdot. Na and E2 .cntdot. (K), respectively, were investigated in the proteolipsomes with particular regard to sidedness of the actions of Na+ and K+. Tryptic digestion of the vesicles reveals the Na+- and K+-stabilized conformations E1 .cntdot. Na and E2 .cntdot. (K) as characterized originally for purified (Na, K)ATPase (Jorgensen, 1975). Controlled trypsinolysis of Tris+-loaded vesicles in a Na+- or K+-containing medium leads to typical biphasic (Na+) or simple exponential (K+) time courses, respectively, for loss of ATP-dependent 22Na uptake (assayed subsequent to the tryptic digestion in the presence of ionophores valinomycin plus FCCP [p-trifluoromethoxycarbonyl cyanide phenylhydrazone]). Tryptic digestion of K+- or Rb+- or Tris+-loaded vesicles suspended in a Na+ medium results only in the biphasic (E1 .cntdot. Na) pattern of loss of ATP-dependent 22Na uptake. ATP-dependent 22Na uptake and 86Rb efflux are reduced by about the same extent following a short tryptic digestion in a Na+-containing medium. Vanadate ions inhibit ATP-dependent 22Na uptake into the vesicles, at low concentrations (K0.5 .apprx. 2 .times. 10-7 M), following pre-incubation together with Mg2+ and K+. K+ in the medium are effective: K+ within the vesicle are not. Na+ in the medium prevent inhibition by vandadate + Mg2+ but do not reverse inhibition in vesicles pre-incubated with vanadate, Mg2+ and K+. The conformational forms E1 .cntdot. Na and E2 .cntdot. (K) are stabilized by Na+ or K+, respectively, bound to sites on the Na/K pump normally facing the cytoplasm. The significance is discussed in relation to the cation transport function of the pump.