The long-term aim of our research is to understand how receptors for neurotransmitters operate in health and disease states. We have chosen the acetylcholine receptor (AChR) at the motor synapse to investigate the essential function of this class of proteins, that of transducing neurotransmitter binding into opening of an intrinsic ion channel. Toward understanding the binding-opening process, we propose to define the structure of the ligand binding site, identify residues that stabilize bound agonist, and identify structures that transmit free energy released by agonist binding to the channel gating apparatus. Our approach is to combine site-directed mutagenesis and expression in mammalian cells with a battery of functional measurements including ligand binding, single channel recording, and protein biochemistry.
AIM (I) will probe the transmitter binding site using the peptide ligand conotoxin M1. We will make mutations in both the AChR and conotoxin to identify pairs of residues that stabilize the toxin-AChR complex.
AIMS (II) and (III) will further define the structure of the binding site by identifying residues in non-alpha subunits of the AChR that confer selectivity of binding of the agonists acetylcholine and epibatidine.
AIM (IV) will identify residues that couple agonist binding to channel gating by making subunit chimeras and point mutations of conserved residues. Subunit association, ligand binding single channel currents will be measured to identify residues underlying inter-subunit communication.
AIM (V) will confirm pathogenicity and delineate structural and functional mechanisms of mutations in human AChR that underlie congenital myasthenic syndromes (CMS). Completion of this aim will contribute to treatment of patients with CMS, as well as provide insights into structure-function relationships of the AChR. Because of their position in the pathway of information flow, neurotransmitter receptors are logical targets for learning and memory processes, pathologic processes and therapeutic drugs. Completion of the aims outlined in this proposal will contribute to treatment and prevention of neuromuscular disorders such as CMS, while the general findings may lead to insights into the operation of members of the superfamily of neurotransmitter receptors.
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