Autosomal dominant polycystic kidney disease is one of the most common inherited diseases in humans. Recently, the two genes responsible for nearly all cases of this disease have bene isolated, PKD1 and PKD2. PKD1 is predicted to contain multiple transmembrane domains and has motifs suggesting that it interacts with the extracellular matrix. PKD2 is predicted to have six transmembrane domains, a topology that is reminiscent of the superfamily of voltage-gated and second messenger-gated ion channels. Moreover, PKD2 has modest, but significant homology to several types of ion channels. The predicted structure of PKD2 has led to the suggestion it may be a cation channel. Nevertheless, the functions of PKD1 and PKD2 remain obscure. To characterize the functions of PKD2, both in vivo and in vitro, this proposal focuses on model experimental organism, the fruitfly Drosophila melanogaster. An opportunity to use this model organism has emerged through the identification of a Drosophila homolog of PKD2 (Dpkd2). The proposed research takes multi-disciplinary approach to the characterization of dPKD2 using a combination of genetic electrophysiological, cell biological, biochemical and molecular approaches.
The first aim of the proposed research is concerned with completing a molecular analysis of dPKD2, including characterization of the spatial distribution of Dpkd2. A second and major aim is to define the role of dPKD2 in vivo. A third goal is to test the hypothesis that dPKD2 is a cation influx channel. The modes of activation, regulation and the biophysical properties of the putative channel will be explored.
A fourth aim i s to characterize the topology and multimerization of Dpkd2. While PKD2 has been proposed to consist of six transmembrane domains, there is currently no experimental evidence to support this model. Finally, the fifth specific aim will be devoted to testing the hypothesis that dPKD2 may also form functional heteromultimeric channels with a related channel, TRP, in vitro and in vivo. Human PKD2 has recently been shown to bind to TRPC in vitro. However, such interactions have not been described in vitro and the functions of the PKD2/TRP association are not known. The long-term goal of the proposed research is to understand the normal roles of PKD2 and the basis for the disease state at the molecular level.
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