Cell Biology of Ion Pumps: Sorting and Function

Na，K泵和胃H，K泵是P型离子转运ATPase家族的成员。虽然这两个泵在结构和机械上有很大的同源性，但它们在亚细胞定位、调节和底物特异性方面有所不同。在前四年的资助期间，我们已经确定了这些分子中每个分子中的结构域，这些结构域在一定程度上解释了这些差异。我们发现，在这些蛋白质的线性序列中广泛分离的基序协作形成构象定义的决定因素，这些决定因素指定泵分布和阳离子选择性。最近，P型肌浆网Ca-ATPase的结构已经在2.6埃单位的分辨率下得到了解决。Na，K和H，K-ATPase与钙泵密切相关，它们的结构几乎可以肯定地反映出这种同源性。我们将利用Ca-ATPase的结构作为指导，产生由Na，K和H，K泵的互补部分组成的新型嵌合多肽。通过评估这些嵌合体的分类和催化性质，我们将能够识别组成这些决定因素的残基，并确定它们在泵蛋白质的三级结构中聚集在一起的机制。我们还将对与Na，K和H，K泵多肽相互作用的蛋白质进行两次杂交筛选，使用这些蛋白质中与Ca-ATPase结构中的自主单元相对应的部分作为诱饵。最后，我们将利用细胞培养系统以及新产生的基因敲除小鼠模型来评估泵结构域和蛋白质-蛋白质相互作用在调节原位离子泵功能和分布中的作用。这些研究将使我们能够确定特定的分子信号和关联是否以及如何可能与胃溃疡疾病和高血压等具有临床意义的病理学相关。

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.