Ligand-dependent effects on the conformational equilibrium of the Na+,K+-ATPase as monitored by voltage clamp...

Voltage clamp fluorometry was used to monitor conformational changes associated with electrogenic partial reactions of the Na(+),K(+)-ATPase after changes in the concentration of internal sodium (Na(+)(i)) or external potassium (K(+)(o)). To probe the effects of the Na(+)(i) concentration on the Na(+) branch of the Na(+),K(+)-ATPase, oocytes were depleted of Na(+)(i) and then loaded with external sodium (Na(+)(o)) using the amiloride-sensitive epithelial sodium channel. The K(+) branch of the Na(+),K(+)-ATPase was studied by exposing the oocytes to different K(+)(o) concentrations in the presence and absence of Na(+)(o) to obtain additional information on the apparent affinity for K(+)(o). Our results demonstrate that lowering the concentration of Na(+)(i) or increasing the amount of K(+)(o) in the external solution shifts the equilibrium toward E(1)/E(1)P. Furthermore, the K(+)(o)-induced relocation toward E(1) occurs at a much lower K(+)(o) concentration when Na(+)(o) is absent, indicating a higher apparent affinity. Finally, voltage-dependent steps associated with the K(+) branch or the Na(+) branch of the Na(+),K(+)-ATPase are affected by the K(+)(o) concentration or the Na(+)(i) concentration, respectively.