Cation-Binding Sites of the Na,K- and H,K-ATPases

Washington University School Of Medicine, Saint Louis MO

Investigators

Abstract

9982901 Blanco The Na,K- and H,K-ATPases belong to a family of ion-motive ATPases known as P-type ATPases. Using the energy from the hydrolysis of ATP, these enzymes are responsible for the active transport of extracellular K+ in exchange for intracellular Na+ or H+. The Na,K-ATPase is critical in maintaining the osmotic balance of the cell, the resting membrane potential of most tissues, and the excitable properties of muscle and nerve cells. In addition, the enzyme plays a primary role in driving the reabsorption of Na+ and water in many epithelia. The H,K-ATPase is essential for the secretion of acid by the stomach and kidney. Structurally, these ion pumps are heterodimers composed of a catalytic a and a glycosylated b subunit. For the Na,K-ATPase various isozymes with distinct functions have been described in different tissues and species. The Na,K- and H,K-ATPases display a high degree of homology in structure and share similar biochemical reaction mechanisms. Despite the progress made in characterizing the structure and function of these ion transporters, uncertainties remain as to the sites of cation-binding and to the structural basis of ion selectivity and cation affinity. The aim of the present research project is to identify the amino acids involved in the cation specificity of the Na,K-ATPase and H,K-ATPase. This will be accomplished by exchanging amino acids that are not shared between the a subunits of the Na,K- and H,K-ATPase and evaluating the effect that the replacements have on the binding of Na+ or H+. These results will provide important information on the structural basis underlying the function of the ion-motive ATPases. In addition, the experiments proposed will be fundamental for further studies of the residues responsible for the differences in cation affinity of the Na,K-ATPase a isoforms.