The structure and function of nicotinic acetylcholine receptors (AChRs) are being investigated. Specific immunosuppressive therapy of the autoimmune response to muscle AChRs which occurs in myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), is also being investigated. One goal is to determine the amount, subunit composition, stoichiometry, and arrangement of neuronal AChR subtypes. This will require expanding our library of subunit-specific monoclonal antibodies (mAbs)and immunoisolating native AChRs. Determining subunit stoichiometry and arrangement will involve expressing cloned AChRs. A second goal is to express a properly assembled, water-soluble extracellular domain of an AChR in amounts suitable for crystallographic studies. We have succeeded in expressing tiny amounts of water-soluble 7 AChR extracellular domains with native ligand binding proteins, but will need to devise methods to increase the efficiency of assembly and increase the amount of protein expressed. This could lead to x-ray crystallographic determination of the structure of an archetypic member of a gene superfamily of wide medical significance. A third goal is to determine the pharmacological and electrophysiological properties of cloned human neuronal AChR subtypes. In addition to expression studies in Xenopus oocytes, this will involve development of a series of permanently transfected cell lines. This should define the functional properties of AChR subtypes, which are the target of nicotine and potential subtype-specific nicotinic drugs now being developed. A fourth goal is to determine the mechanisms by which chronic exposure to nicotine alters the amounts and functional properties of cloned human neuronal AChRs. These studies should reveal some of the molecular mechanisms by which nicotine produces its and huge medical impact, while also providing insights on possible effects of nicotinic agonists being developed as drugs. A fifth goal is to determine the location of AChR subtypes. This will involve providing our expanded library of AChR-subunit specific mAbs to collaborators. A sixth goal is to develop a practical specific immunosuppressive therapy for EAMG. This will involve inducing tolerance through administration of bacterially-expressed human muscle AChR subunits. It is hoped that this will lead to a specific therapy for MG.
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