All animal cells require maintenance of transmembrane gradients for Na+ and K+ ions by the Na/K pump, a plasma membrane P-type ATPase. Pathophysiologically, the Na/K pump is the target specifically inhibited by cardiotonic steroids, drugs traditionally used for the treatment of heart failure. Also, mutations in the genes encoding for two Na/K pump isoforms have been linked to migraine and Parkinsonism. The long-term goal of our laboratory is to understand the relationship between the Na/K pump structure, its function, and its multiple roles in cardiovascular and neurological diseases. All P-type ATPases alternate between two major conformers E1 and E2. The molecular forces that stabilize intermediate states in the cycle must arise as a consequence of a set of state-specific residue-residue and residue-ligand interactions. The main research goal of this AREA project is to identify and characterize energetically and mechanistically relevant interactions between pairs of residues and between residues and ions within the Na/K pump. To address this problem we use mutagenesis, heterologous expression of Na/K pumps in Xenopus oocytes, voltage clamp (two electrode voltage clamp, cut-open oocyte and patch clamp), chemical modification of engineered cysteine residues and computational chemistry to address two independent and interrelated aims, which we propose on the basis of extensive preliminary work: 1) To identify and characterize critical conformation-specific interactions that stabilize Na/K pump conformations, 2) to elucidate the mechanisms of ion-induced conformational changes. Considering the overwhelming evidence that the rate of E1 - E2 conversion underlies the molecular mechanism of this critical transporter, the work proposed here will be crucial to understanding and resolving the etiology of clinically relevant problems. Specifically, dysfunction of the Na,K-ATPase has been attributed to hypertension, congestive heart failure, familial hemiplegic migraine, and polycystic kidney disease.