There are two components to this Core, electrophysiology ancUmaging. I. The electrophysiology component of this Core will provide resources to qualitatively screen novel conopeptides to identify their target as well as quantitatively assess the functional activities of conopeptides against known ion channel targets. Assays at three levels will be performed, where each successive level involves decreased scope and increased specificity: 1) Extracellular recording from amphibian and rodent skeletal muscle and peripheral nerves (including motor, sensory, and sympathetic nerves) in isolated tissue preparations will be used to obtain a global assessment of a conopeptide's activity. 2) Whole-cell voltage clamping of dissociated neurons will be used to identify the type of the channel affected by a given conopeptide (e.g., voltage-gated Na vs. K channels). 3) The specific channel isotype targeted by the conopeptide will be pinpointed by examining a conopeptide's effect on cloned channels expressed in Xenopus oocytes. Levels 2 and 3 will also allow the mechanism of conopeptide-action to be addressed. II. The imaging component of the Core will be used to identify and investigate the sites of conopeptide binding and action at the tissue, cellular, and molecular levels by light microscopy. There are three facets to this endeavor. 1) Derivatize conopeptides that have known target-specificities with fluorescent reporter groups. Characterize the adducts'binding affinities, target specificities, and functional properties to see if any of these are altered by the presence of the reporter group. 2) Use of the labeled conopeptides to identify the locations of their binding sites in isolated tissues and in brain and spinal cord slices by confocal fluorescence microscopy. 3) Examine the interactions of conopeptides with their target channels at the molecular level by single molecule imaging using total internal reflection (or evanescent wave) fluorescence microscopy (TIRFM). Here, fluorescently labeled conopeptides will be used in conjunction with Xenopus oocytes expressing cloned channels and subsequently dissociated cells, a) The number of conopeptide molecules bound to a single channel will be determined by counting bleaching-steps. b) For nAChRs that are permeable to Ca++, functional consequence of conopeptide-binding can be concurrently monitored by calcium imaging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM048677-18
Application #
8145712
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
18
Fiscal Year
2010
Total Cost
$215,114
Indirect Cost
Name
University of Utah
Department
Type
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Yan, Yijin; Peng, Can; Arvin, Matthew C et al. (2018) Nicotinic Cholinergic Receptors in VTA Glutamate Neurons Modulate Excitatory Transmission. Cell Rep 23:2236-2244
Hone, Arik J; McIntosh, J Michael (2018) Nicotinic acetylcholine receptors in neuropathic and inflammatory pain. FEBS Lett 592:1045-1062
Hone, Arik J; Talley, Todd T; Bobango, Janet et al. (2018) Molecular determinants of ?-conotoxin potency for inhibition of human and rat ?6?4 nicotinic acetylcholine receptors. J Biol Chem 293:17838-17852
Banala, Sambashiva; Arvin, Matthew C; Bannon, Nicholas M et al. (2018) Photoactivatable drugs for nicotinic optopharmacology. Nat Methods 15:347-350
Hone, Arik J; Servent, Denis; McIntosh, J Michael (2018) ?9-containing nicotinic acetylcholine receptors and the modulation of pain. Br J Pharmacol 175:1915-1927
Espino, Samuel S; Robinson, Samuel D; Safavi-Hemami, Helena et al. (2018) Conopeptides promote itch through human itch receptor hMgprX1. Toxicon 154:28-34
Richter, Katrin; Sagawe, Sabrina; Hecker, Andreas et al. (2018) C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation. Front Immunol 9:1604
Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin et al. (2018) ?-Nicotinamide Adenine Dinucleotide (?-NAD) Inhibits ATP-Dependent IL-1? Release from Human Monocytic Cells. Int J Mol Sci 19:
Peng, Can; Yan, Yijin; Kim, Veronica J et al. (2018) Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission. Eur J Neurosci :
Chen, De-Jie; Gao, Fen-Fei; Ma, Xiao-Kuang et al. (2018) Pharmacological and functional comparisons of ?6/?3?2?3-nAChRs and ?4?2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line. Acta Pharmacol Sin 39:1571-1581

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