Cell Biology of Ion Pumps: Sorting and Function

点击翻译按钮获取中文摘要

The Na,K and gastric H,K pumps are members of the P-type family of ion transporting ATPases. While these two pumps share a great deal of structural and mechanistic homologies, they differ in their subcellular localizations, regulation, and substrate specificities. During the previous four year funding period we have identified domains in each of these molecules that account in part for these differences. We have found that motifs widely separated in these proteins' linear sequences collaborate to form conformationally-defined determinants that specify pump distribution and cation selectivity. Recently, the structure of the P-type sarcoplasmic reticulum Ca-ATPase has been solved at 2.6 Angstrom units resolution. The Na,K and H,K-ATPases are closely related to the Ca pump, and their structures are almost certain to reflect this homology. We will use the structure of the Ca-ATPase as a guide in generating novel chimeric polypeptides composed of complementary portions of the Na,K and H,K pumps. By assessing the sorting and catalytic properties of these chimeras, we will be able to identify the residues that comprise these determinants and to define the mechanism through which they are brought together in the tertiary structures of the pump proteins. We will also conduct two hybrid screens for proteins that interact with the Na,K and H,K pump polypeptides, using as baits portions of these proteins that correspond to autonomous units in the Ca-ATPase structure. Finally, we will utilize cell culture systems as well as newly generated knockout mouse models to assess the roles of pump domains and protein- protein interactions in regulating the function and distributions of ion pumps in situ. These studies will allow us to determine whether and how specific molecular signals and associations might be related to such clinically significant pathologies as gastric ulcer disease and hypertension.