EXCEED THE SPACE PROVIDED. The 5-Hydroxytryptamine3 (5-HT3) receptor, which may be composed of homomers of A subunits or heteromers of A and B subunits, is the least studied member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. In addition to its role in mediating synaptic transmission in the nervous system, it also regulates gastrointestinal motility and the vomiting reflex. The recently published crystal structure of the :acetylcholine binding protein (Brejc et al., 2001) has identified the generalized structure of the N-terminal domains of this superfamily, but the precise three-dimensional configuration of the ligand recognition site and the residues involved in mediating ion channel activation are unknown. The goal of this proposal is to map amino acid residues that contribute to ligand recognition and model their spatial configuration. Mouse and human 5-HT3A receptors possess 84% identity at the amino acid level, yet have differential sensitivities to i numerous drugs that bind to the ligand recognition site, such as d-tubocurarine (curare) and 3-(2-hydroxy, 4- methoxy-benzylidene)-anabaseine (2-OHMBA). The addition of the B subunit to the A subunit further alters ipotency of these compounds. Mouse-human and human-mouse chimeras will be constructed and expressed in Xenopus oocytes. Domains responsible for the change in drug action will be assessed with two-electrode voltage clamp electrophysiological recordings. Individual amino acids will then be identified for their roles in conferring sensitivity. Structure-activity relationships of key moieties in the substituted 3-benzylidene- anabaseine analogs with identified residues will be assessed. Thermodynamic mutant cycle analysis will pinpoint the specific point of contact of the key drug moiety with the identified amino acid residue in the ligand binding domain. 5-HTaAreceptors with C-terminal hexa-histidine tags will be expressed and purified. Receptor will then be photolabeled with [3H]-5-HT and [14C]-2-OHMBA, with and without azido side-chains, and radiolabeled residues will be identified through sequencing. Amino acids identified with photoaffinity labeling and electrophysiological experiments, along with the structures of 5-HT, curare, and benzylidene-anabaseine analogs, will serve as a template for molecular modeling studies of the agonist-recognition site. PERFORMANCE SITE ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS043438-03
Application #
6822013
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Silberberg, Shai D
Project Start
2002-12-01
Project End
2006-11-30
Budget Start
2004-12-01
Budget End
2005-11-30
Support Year
3
Fiscal Year
2005
Total Cost
$233,724
Indirect Cost
Name
University of North Texas
Department
Pharmacology
Type
Other Domestic Higher Education
DUNS #
110091808
City
Fort Worth
State
TX
Country
United States
Zip Code
76107
Machu, Tina K (2011) Therapeutics of 5-HT3 receptor antagonists: current uses and future directions. Pharmacol Ther 130:338-47
Sanghvi, Mitesh; Hamouda, Ayman K; Davis, Margaret I et al. (2009) Hydrophobic photolabeling studies identify the lipid-protein interface of the 5-HT3A receptor. Biochemistry 48:9278-86
Hamouda, Ayman K; Sanghvi, Mitesh; Sauls, Daniel et al. (2006) Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 45:4327-37