About the Lab
Allosteric mechanisms of ligand-gated ion channels: ionic selectivity, agonist-mediated activation, and conformational changes are determined with LRET, stopped-flow fluorescence and ligand-binding energetics. Computational drug discovery is used to develop lead compounds for nicotine replacement therapy.
Our research is centered on understanding the molecular mechanisms of ligand gated ion channels, particularly 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
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 fluorescent analogs 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 transitions are investigated by measurement of protein movement using lanthanide-based luminescent energy transfer methods (LRET). In particular, we synthesize lanthanide-based fluorophores for precise distance measurements of conformational changes and apply this approach to the nicotinic acetylcholine receptor.