Our research is centered on understanding the molecular mechanisms of ligand gated ion channels, paticularly the nicotinic acetylcholine receptor. These channels mediate chemical signal transduction in the nervous system. The nicotinic receptor is located at the neuromuscular junction where it receives signals from motor neurons and initiates electrical impulses in the muscle. We focus on the structural features and conformational changes that regulate channel function. Normal binding and conformational stability are critical for nicotinic acetylcholine receptor function at the neuromuscular junction; abnormalities and mutations lead to various diseases, including congenital myasthenia gravis. Conformational changes are regulated by the binding of small ligands to specific binding sites that are also the targets of many toxins such as d-tubocurarine, alpha-bungarotoxin, and alpha-conotoxins.
Current research efforts in the lab comprise three general areas: structure-activity of ligand binding, kinetic analysis of conformational transitions, and determination of structural changes upon binding and conformational changes. Toxins provide powerful tools for investigating the linkage between binding and channel opening. Altering the structure of the toxins by making analogs, allows us to manipulate their activity. By coupling this approach with site-directed mutagenesis at ligand binding sites, we delineate the pathway of conformational changes at the acetylcholine binding sites that lead to channel opening.
Kinetic analysis of ligand binding is carried out using a fluorescent analog of acetylcholine. Stopped-flow fluorescence determination of the kinetics of binding gives us a model of the conformational transitions of the receptor, how many conformations it can adopt, and the pathway taken during normal activation. We analyze kinetic data using software written in the lab for general modeling of complex kinetic mechanisms.
Structural changes that accompany conformational transistions are investigated by measurement of protein movement using fluorescent energy transfer methods. In particular, we are developing lanthanide-based fluorophores. These fluorophores allow fluorescence lifetime measurements that give precise energy transfer distance measurements. Such measurements are also used to determine fundamental aspects of ligand-binding kinetics, in particular, the influence of electrostatic surface potentials on the rate of binding of charged ligands.