P2X receptors are transmitter-gated ion channels activated by extracellular ATP. The distribution, topology, pharmacology, and physiology of the seven members of the family (P2X1.7) are well documented. By contrast, the signal transduction pathway is poorly understood. We hypothesize that activation of the receptor involves the following steps: First, ATP binds to a site on the extracellular surface of the protein complex. Second, occupation of this site results in a change in the shape of the channel pore that permits ion conduction to occur. Third, Na+ and Ca2+flow down their electrochemical gradients and into the cell. Fourth, the inward flux of Na+ renders the cell hyperexcitable by depolarizing the membrane and the inward flux of Ca2+ triggers numerous cell-specific sequella such as muscle contraction, neurotransmitter release, and sensation. An additional fifth step occurs in some receptor subtypes (P2X2, 4.7) when ATP is applied for more than a few seconds; here, the narrowest part of the pore dilates to a size that allows larger cations like N-methyI-D-glucamine (NMDG) and the cationic cyanine dye, YO-PRO-1, to permeate the channel. The functional sequella of dilation include blebbing, microvesiculation, and cell death, actions that may involve intra- and/or inter-molecular interactions of the intracellular C-terminal tail of the receptor. The goal of the experiments outlined in this proposal is to provide a better description of the dynamics of P2X channels during gating, conduction, and pore dilation. In the first aim, we use several techniques to quantify ion flux through homomeric and heteromeric P2X receptors, and we compare these fluxes to those seen in other members of the transmitter-gated ion channel superfamily. Further, we use site-directed mutagenesis to identify domains within the pore that regulate permeability and flux across the surface membrane. In the next two aims, we study the molecular motions of the channel during gating and dilation using two different techniques. In the first set of experiments, an array of cysteine-substituted mutants and thiol-reactive benzophenones will be used to map the position of residues within the transmembrane segments before, during, and after applications of ATP. In the second set of experiments, fluorescence resonance energy transfer (FRET) will be used to determine intra- and inter-molecular distances in the absence and presence of ATP.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070925-03
Application #
7047793
Study Section
Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
Program Officer
Chin, Jean
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
3
Fiscal Year
2006
Total Cost
$323,687
Indirect Cost
Name
Saint Louis University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Sáez-Orellana, Francisco; Fuentes-Fuentes, María C; Godoy, Pamela A et al. (2018) P2X receptor overexpression induced by soluble oligomers of amyloid beta peptide potentiates synaptic failure and neuronal dyshomeostasis in cellular models of Alzheimer's disease. Neuropharmacology 128:366-378
Han, Junbin; Liu, Hui; Liu, Chunling et al. (2017) Pharmacologic characterizations of a P2X7 receptor-specific radioligand, [11C]GSK1482160 for neuroinflammatory response. Nucl Med Commun 38:372-382
Kracun, Sebastian; Chaptal, Vincent; Abramson, Jeff et al. (2010) Gated access to the pore of a P2X receptor: structural implications for closed-open transitions. J Biol Chem 285:10110-21
Scatizzi, John C; Mavers, Melissa; Hutcheson, Jack et al. (2009) The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages. Eur J Immunol 39:820-5
Samways, Damien S K; Harkins, Amy B; Egan, Terrance M (2009) Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry. Cell Calcium 45:319-25
Young, Mark T; Fisher, James A; Fountain, Samuel J et al. (2008) Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy. J Biol Chem 283:26241-51