Extracellular adenosine 5'-triphosphate (ATP) affects a wide variety of cells via purinergic membrane receptors. Several classes of purinergic receptors have been identified based on agonist specificity and intracellular signaling pathways. One class, the P2x receptor, forms ATP-gated, calcium-permeable, cation-selective channels. Although there have been no published reports about the expression of ionotropic P2x receptors in renal cells, the applicant has obtained preliminary data describing both functional and molecular evidence of the expression of these receptors on cultured renal epithelial cells (LLC-PK1). Defining the interactions of ATP with its receptors on renal epithelial cells will lead to a better understanding of the mechanisms by which extracellular nucleotides regulate renal epithelial cell transport and physiology. Therefore, the studies described in this proposal will address the hypothesis that extracellular ATP affects renal epithelial cells via interaction with an ionotropic receptor of the P2x class. In addressing this hypothesis, the following Specific Aims are proposed.
Specific Aim Number 1: Further characterize, pharmacologically and functionally, the purinergic receptor in LLC-PK1 cells.
Specific Aim Number 2: Isolate and determine the sequence of a full length of cDNA for the P2x receptor in LLC-PK1 cells.
Specific Aim Number 3: Determine the functional characteristics of the cloned P2x receptor. The applicant will employ whole cell and single channel patch clamp techniques, fluorescence measurements of intracellular calcium, molecular cloning, and expression in Xenopus oocytes to accomplish these aims. These studies should provide a basis for future studies aimed at determining the mechanisms whereby extracellular nucleotides affect renal epithelial cell function in human health and disease.
