Developing an understanding of the molecular mechanisms that underlie synaptic communication, and hence form the basis of learning, memory, and sensory perception, stands as one of the central challenges of modern science. In this proposal, the focus is on one large family of neuroreceptors that plays a central role in this process, the ligand-gated ion channels (LGIC). These proteins mediate fast synaptic transmission, and they are the targets of therapeutic approaches to Alzheimer's disease, Parkinson's disease, schizophrenia, stroke, learning and attention deficits, and drug addiction. A major goal of this work is to develop an understanding of the precise chemical interactions that lead to specific and potent drug-receptor interactions, including both drugs of therapeutic value and drugs of abuse. This will be of considerable value in developing new pharmaceuticals with improved selectivities and potencies. Binding of a drug to an LGIC induces a large structural change in the protein that leads to the opening of an ion channel within the protein, a profound signaling event for the cell. This """"""""gating"""""""" process is central to the function of the receptors, and it will be another focus of investigation. LGICs and other neuroreceptors are complex, multisubunit, integral membrane proteins. As such, the powerful tools of structural biology - x-ray crystallography and NMR spectroscopy - are not readily applicable. This work makes use of the in vivo nonsense suppression method for unnatural amino acid incorporation into proteins expressed in living cells. This approach allows almost limitless modification of the receptors. Subtle changes can be made to reveal key binding interactions. More dramatic changes introduce biophysical tools to probe structure and function. When combined with the power of electrophysiology, unnatural amino acid mutagenesis becomes a powerful, broadly applicable tool for unraveling the critical features of these central players in molecular neurobiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37NS034407-14
Application #
7665256
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Silberberg, Shai D
Project Start
1995-07-01
Project End
2011-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
14
Fiscal Year
2008
Total Cost
$414,564
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Mosesso, Richard; Dougherty, Dennis A (2018) A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors. J Biol Chem 293:2903-2914
Post, Michael R; Lester, Henry A; Dougherty, Dennis A (2017) Probing for and Quantifying Agonist Hydrogen Bonds in ?6?2 Nicotinic Acetylcholine Receptors. Biochemistry 56:1836-1840
Rienzo, Matthew; Rocchi, Angela R; Threatt, Stephanie D et al. (2016) Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors. J Biol Chem 291:6272-80
Davis, Matthew R; Dougherty, Dennis A (2015) Cation-? interactions: computational analyses of the aromatic box motif and the fluorination strategy for experimental evaluation. Phys Chem Chem Phys 17:29262-70
Henderson, Brandon J; Lester, Henry A (2015) Inside-out neuropharmacology of nicotinic drugs. Neuropharmacology 96:178-93
Marotta, Christopher B; Lester, Henry A; Dougherty, Dennis A (2015) An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in ?7 Nicotinic Acetylcholine Receptors. Chem Biol 22:1063-73
Post, Michael R; Limapichat, Walrati; Lester, Henry A et al. (2015) Heterologous expression and nonsense suppression provide insights into agonist behavior at ?6?2 nicotinic acetylcholine receptors. Neuropharmacology 97:376-82
Miles, Timothy F; Lester, Henry A; Dougherty, Dennis A (2015) Allosteric activation of the 5-HT3AB receptor by mCPBG. Neuropharmacology 91:103-8
Limapichat, Walrati; Dougherty, Dennis A; Lester, Henry A (2014) Subtype-specific mechanisms for functional interaction between ?6?4* nicotinic acetylcholine receptors and P2X receptors. Mol Pharmacol 86:263-74
Dougherty, Dennis A; Van Arnam, Ethan B (2014) In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool. Chembiochem 15:1710-20

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