Ligand-Dependent Effects on the Conformational Equilibrium of the Na+,K+-ATPase As Monitored by Voltage Clamp Fluorometry

Abstract

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 E1/E1P. Furthermore, the K+o-induced relocation toward E1 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.