Structures of members of the ligand gated ion channel gene superfamily which exhibit nicotinic pharmacological properties are being studied. This includes acetylcholine receptors (AChRs) from muscles and nerves as well as neuronal proteins of unknown function which bind a bungarotoxin (alphaBgtBPs). The autoimmune response to muscle AChRs which occurs in myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG) is also being studied. Long-term Objectives and Medical Significance: Goals include determining: 1)structures of the AChR and alphaBgtBP subtypes; 2) how they function; 3) how they are assembled; 4) where they are located; and 5) what their functional roles are. This should help explain nicotinic synaptic transmission and pharmacology at a molecular level, reveal new functional roles for neuronal AChRs and alphaBgtBPs, permit pharmacological studies on human AChR and alphaBgtBP subtypes expressed in cell lines, help explain the mechanisms by which nicotine is addictive to smokers, and help explain AChR loss in Alzheimer's and Parkinson's diseases. Goals for studies of MG will take advantage of structural studies of muscle AChRs to: 1) precisely define the structure of the main immunogenic region (MIR), and 2) provide a specific immunosuppressive therapy for MG.
Specific Aims and Methods: The subunit compositions of immunoaffinity purified neuronal AChR and alphaBgtBP subtypes are being analyzed biochemically; cDNAs encoding these subunits are being identified; and finally, proteins synthesized from combinations of subunit cDNAs thought to comprise the native proteins are being compared with the native proteins. We are studying AChRs from Torpedoes, chickens, and other species' as well as the equivalent human proteins and cDNAs. Using subunit-specific probes, subtypes of neuronal AChRs and alphaBgtBPs are being localized histologically to particular cells and subcellular structures. The transmembrane orientation of AChR subunit polypeptide chains, and the order in which the subunits are organized around the central cation channel will be studied. In addition to continuing attempts to obtain crystals of intact muscle AChRs purified from Torpedo, we will express simplified proteins from cDNAs that may be easier to crystalize including: 1) the extracellular domain of human alphal subunits, 2) homo-oligomeric alphaBgtBP, and 3) neuronal AChRs. Assembly of subunits to form intact AChRs and alphaBgtBPs will be studied using AChRs in cell lines and subunits expressed in various combinations The amino acids critical to the recognition of the MIR on the a subunits of human muscle AChRs will be investigated by in vitro mutagenesis and expression followed by binding studies with MG patient autoantibodies. A small soluble peptide mimotope which models the amino acids recognized by autoantibodies to the MIR in its native conformation will be sought by screening a random peptide library expressed as a fusion protein. If either a mimotope or a soluble expressed a subunit extracellular domain peptide with high affinity for autoantibodies to the MIR can be prepared, these will be used to target toxins to specifically kill autoimmune lymphocytes in EAMG and MG.
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