Ligand-gated ion channels are critical to brain function. These proteins mediate rapid signaling from one neuron to the next, by opening ion selective pores in the surface membrane in response to the binding of neurotransmitter, and thus eliciting ion flows that cause electrical excitation or inhibition. When ligand-gated channels spend too much or too little time open, the brain cannot process information correctly. Furthermore, alterations in ligand-gated channel activity can result in overt neurological disease, including epilepsy and neurodegenerative diseases. It is thus important to understand the molecular mechanisms that allow channels to open properly. The focus of the work described here is on a class of channels gated by extracellular ATP, which are referred to as P2X receptors. Genes encoding seven different P2X receptors are expressed in the mammalian brain. Understanding the role of specific P2X receptors in normal brain function and in brain disease has been impeded by the absence of subunit specific ligands. In particular, the P2X2 subunit is widely expressed in the brain, but its role is unclear. In Goal 1 we will use a novel random mutagenesis strategy to gain information about the structure and function of P2X receptors. This approach should generate a large number of new mutant receptors that could not be identified by any other strategy. In particular, these experiments will provide important new information about the ATP binding site, which will be of tremendous use in developing new, more selective agents. In Goal 2, we will explore the mechanism by which ATP is able to open P2X2 receptor channels in wild type and mutant receptors in detail using single channel recording. These single channel recordings will allow us to test distinct models of receptor activation. Understanding how P2X2 channels open and close is potentially of great significance for understanding brain injury. A great deal of evidence suggests that overactivity of NMDA-class glutamate receptors is a major cause of brain injury. Like NMDA receptors, P2X2 receptors form calcium permeable channels that are modulated by low pH and elevated Zn. However, these modulators inhibit current through NMDA receptors, but potentiate current through P2X2 receptors. Many neurons co-express NMDA and P2X2 receptors. Thus the ratio of P2X2 receptors to NMDA receptors may be a key determinant of whether changes in the brain environment following seizures or ischemia result in hyper or hypoexcitability. This has important ramifications for which neurons are susceptible to damage as a result of these conditions, and for strategies to try to prevent this damage.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-4 (01))
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Talley, Edmund M
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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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
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
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
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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