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 examine the essential function of this class of proteins: binding of neurotransmitter triggering opening of an intrinsic ion channel. Toward understanding this binding-triggering process, we propose to (i) identify residues in a and non-a subunits that stabilize ACh when bound to open and closed states of the channel, (ii) determine how the AChR maintains uniform channel gating kinetics, (iv) determine mechanistic consequences of mutations underlying congenital myasthenic syndromes (CMS), (iv) determine whether conserved residues bounding putative secondary structures in the major extracellular domain contribute to AChR activation and (v) determine structural features of the major extracellular domain of the AChR. The approach combines site-directed mutagenesis and expression in mammalian cells, single channel recording and kinetic analysis, measurements of ligand binding and protein biochemistry. Completion of the proposed studies will advance our understanding of synaptic transmission and drug action at motor endplates, facilitate treatment of neuromuscular disorders such as CMS, while the general findings will provide insight into structure function relationships for other members of the neurotransmitter receptor superfamily.
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