MOLECULAR AND FUNCTIONAL ANALYSIS OF ATP RECEPTORS

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Evidence accumulated over the past three decades supports the role of adenosine-tripphosphate (ATP) as a neurotransmitter/neuromodulator in many adrenergic, cholinergic and non-adrenergic/non/cholinergic pathways. Its role as a chemical mediator of intercellular communication has been established in tissues such as the central and peripheral nervous systems, endocrine glands, heart and smooth muscle. However, the mechanisms which mediate ATP's role as a chemical signal is poorly understood. A primary reason for this is the presence of multiple receptor subtypes in most tissues coupled with a lack of subtype-selective agonists and antagonists. Additionally, in some tissues there are high levels of cell-surface enzymes (ecto-ATPases) that can quickly degrade many of the ATP analogues used for receptor classification, resulting in further complications. In order to clearly understand the role of ATP-mediated transmission, it is essential to define the pharmacological and physiological properties of ATP receptors. Currently, ATP receptors fall into one of two classifications, P2Y and P2X receptors. The focus of this grant are the ATP receptors which belong to the P2X category. These receptors are ligand-gated channels that are permeable not only to monovalent cations such as Na+ and K+, but also to Ca2+. In this project, we will circumvent the difficulties associated with investigating multiple receptor subtypes in their native settings through the use of recombinant receptor expression in mammalian cells. This approach allows for the expression of individual receptor subtypes in a controlled environment, thus permitting detailed characterization of the basic properties of a receptor subunit. We have embarked on the investigation of P2X receptors at the molecular and functional level using a compare and contrast methodology. In this study we will elucidate the stoichiometry of receptor assemblies, elucidate the domains involved in subunit interactions and identify amino acid residues that participate in agonist (binding and gating of the channel. These experiments will provide essential information regarding P2X receptor function and the role of ATP as a neurotransmitter in the central and peripheral nervous systems. The overall goal of this project is to provide a clearer understanding of the pharmacological and physiological properties associated with specific P2X receptor subtypes.