Structure-function studies of agonist binding to the muscle-type nicotinic acetylcholine receptor and the development of a trifunctional non-competitive antagonist suitable for activity-dependent profiling

The muscle-type nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel required for fast synaptic transmission at the neuromuscular junction. It is the archetype of the Cys-Loop superfamily of receptors and a prototypic allosteric protein. The muscle-type AChR has two distinct transmitter binding sites found in the extracellular ligand-binding domain. When acetylcholine binds these sites, a series of still unresolved conformational changes occur, leading to opening of the transmembrane pore over 40 A distant from the binding sites. High resolution structures of the intact receptor and the acetylcholine binding protein have provided greater insight into the structural basis of the allosteric mechanism coupling agonist binding and pore opening. However, comprehensive models of the agonist-bound receptor in its closed and open states are still not available. In particular, the details describing the conformation of binding site residues and the dynamics of their interactions with agonists and competitive antagonists are still under investigation. These details are of particular importance to the design of AChR agonists, partial agonists, and competitive antagonists which may have therapeutic potential for treating neuromuscular and neurological pathologies. Using single-channel electrophysiology we investigated details of the agonist-bound open-state transmitter binding sites. Using a series of structurally related organic cations, we observed a structure-activity relationship that suggests cation-n binding interactions are important for open-state affinity. We also conducted a structure-function study to measure kinetic and thermodynamic differences in agonist binding to the two different transmitter binding sites in both the closed and open states. We observed that the two binding sites have unequal affinities for the agonist choline in the closed state and equal affinities in the open state. The state-dependent difference in affinities suggests that binding determinants from the a subunits predominantly determine open-state choline affinity at each site.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Tantama, Mathew C

Advisor dc:contributor.advisor

• Stuart S. Licht.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/46035

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/46035