The P2X proteins are ATP gated channels that depolarize cells and also allow calcium to enter. P2X receptors are expressed in virtually every tissue, including neurons and glia of the central and peripheral nervous system, smooth, skeletal and cardiac muscle, cochlear hair cells, platelets, most classes of white blood cells, hepatocytes, and endothelial cells in the lung and gastrointestinal tract. The importance of members of this gene family for normal physiology is apparent from the range of phenotypes that are seen in their absence, Mice in which specific P2X receptors are knocked out show dysfunction in pain perception, ability to void the bladder, gut motility, neuronal control of ejaculation, the ability of the nervous system to monitor the oxygen level in the blood, the ability to fight bacterial infection, and blood clotting. A major problem in the purinergic receptor field is the limited specificity of agonists and antagonists that can be used to alter ATP signaling in vivo. The goal of the experiments described here is to better characterize the molecular mechanisms that allow ATP and allosteric modulators to open P2X receptor channels. The results of these studies should facilitate the development of agents tha't act more specifically on particular receptors. We will use electrophysiological, biochemical, and molecular approaches to study receptors bearing complementary mutations in adjacent or non-adjacent subunits.
The specific aims are: Goal 1- To test whether the zinc binding sites that modulate channel activity in P2X2, P2X3, and P2X4 receptors are within or between subunits, and to define residues that participate in these binding sites. We will also define our understanding about the mechanisms by which zinc promotes channel opening in P2X2 receptors. Goal 2 - To test whether the ATP binding site of P2X receptors is within or between subunits and to define additional residues that are exposed in the ATP binding pocket. These experiments will also test the number of molecules of ATP that must be bound in order to open a channel. Goal 3 - To define the molecular movements that are a consequence of zinc or ATP binding to P2X2 receptors. These experiments are of particular relevance to making progress in understanding and treating pain associated with tissue injury, as P2X2 and P2X3 receptors have been implicated as playing essential roles as sensing the damage and signaling the central nervous system. Having a better understanding of the structure of these receptors should allow the development of new treatments for this type of pain, and so greatly ease the suffering of individuals with burns and other injuries that produce persistent pain.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS039196-08
Application #
7534313
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Stewart, Randall R
Project Start
2001-03-01
Project End
2010-11-30
Budget Start
2008-12-01
Budget End
2010-11-30
Support Year
8
Fiscal Year
2009
Total Cost
$317,111
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Punthambaker, Sukanya; Hume, Richard I (2014) Potent and long-lasting inhibition of human P2X2 receptors by copper. Neuropharmacology 77:167-76
Punthambaker, Sukanya; Blum, Jacob A; Hume, Richard I (2012) High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism. J Biol Chem 287:22099-111
Dellal, Shlomo S; Hume, Richard I (2012) Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP. PLoS One 7:e47147
Friday, Sean C; Hume, Richard I (2008) Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors. J Neurochem 105:1264-75
Tittle, Rachel K; Hume, Richard I (2008) Opposite effects of zinc on human and rat P2X2 receptors. J Neurosci 28:11131-40
Moffatt, Luciano; Hume, Richard I (2007) Responses of rat P2X2 receptors to ultrashort pulses of ATP provide insights into ATP binding and channel gating. J Gen Physiol 130:183-201
Tittle, Rachel K; Power, Jamila M; Hume, Richard I (2007) A histidine scan to probe the flexibility of the rat P2X2 receptor zinc-binding site. J Biol Chem 282:19526-33
Nagaya, Naomi; Tittle, Rachel K; Saar, Nir et al. (2005) An intersubunit zinc binding site in rat P2X2 receptors. J Biol Chem 280:25982-93
Cui, Wilson W; Low, Sean E; Hirata, Hiromi et al. (2005) The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS. J Neurosci 25:6610-20
Clyne, J D; Brown, T C; Hume, R I (2003) Expression level dependent changes in the properties of P2X2 receptors. Neuropharmacology 44:403-12

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