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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
3R01NS035534-04A2S1
Application #
6353814
Study Section
Special Emphasis Panel (ZRG1 (01))
Program Officer
Michel, Mary E
Project Start
1996-09-15
Project End
2004-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
4
Fiscal Year
2000
Total Cost
$37,000
Indirect Cost
Name
Saint Louis University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Kucenas, S; Li, Z; Cox, J A et al. (2003) Molecular characterization of the zebrafish P2X receptor subunit gene family. Neuroscience 121:935-45
Diaz-Hernandez, Miguel; Cox, Jane A; Migita, Keisuke et al. (2002) Cloning and characterization of two novel zebrafish P2X receptor subunits. Biochem Biophys Res Commun 295:849-53
Haines, W R; Migita, K; Cox, J A et al. (2001) The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel. J Biol Chem 276:32793-8
Migita, K; Haines, W R; Voigt, M M et al. (2001) Polar residues of the second transmembrane domain influence cation permeability of the ATP-gated P2X(2) receptor. J Biol Chem 276:30934-41
Haines, W R; Voigt, M M; Migita, K et al. (2001) On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor. J Neurosci 21:5885-92
Cox, J A; Barmina, O; Voigt, M M (2001) Gene structure, chromosomal localization, cDNA cloning and expression of the mouse ATP-gated ionotropic receptor P2X5 subunit. Gene 270:145-52
Egan, T M; Cox, J A; Voigt, M M (2000) Molecular cloning and functional characterization of the zebrafish ATP-gated ionotropic receptor P2X(3) subunit. FEBS Lett 475:287-90
Haines, W R; Torres, G E; Voigt, M M et al. (1999) Properties of the novel ATP-gated ionotropic receptor composed of the P2X(1) and P2X(5) isoforms. Mol Pharmacol 56:720-7
Torres, G E; Egan, T M; Voigt, M M (1999) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem 274:6653-9
Torres, G E; Egan, T M; Voigt, M M (1998) N-Linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor. Biochemistry 37:14845-51

Showing the most recent 10 out of 13 publications