This research program is organized around 4 specific aims concerned with the structure and function of human neuronal nicotinic receptors (AChRs) and the effects of nicotine on these AChRs. These studies of neuronal AChRs are intended to increase understanding of their synthesis, physiological properties, their complex responses to nicotine and potential nicotinic drugs, and their roles in neurological diseases including Parkinson's, Alzheimer's, schizophrenia, and autoimmune dysautonomias.
Aim 1 is to investigate the variable subunit stoichiometry of alpha4beta2 AChRs. In our cell line, these AChRs are found both in an (alpha4)2(beta2)3 and an (alpha4)3(beta2)2 stoichiometry which differ substantially in their properties. By expressing linked alpha4 and beta2 subunits alone or in combination with free subunits it is possible to make linked AChR pairs with one of each stoichiometry or monopentamers of either stoichiometry. We will search for both stoichiometries in neurons. We will use linked subunits to prepare alpha4beta2, alpha4beta2alpha5, alpha4alpha6beta2 and other subunit combinations in defined stoichiometries and subunit arrangements and determine their physiological properties.
Aim 2 is to determine the effects of prolonged exposure to nicotine and nicotinic drugs on the function of alpha3, alpha4, alpha6, and alpha7 AChR subtypes, and to determine the mechanisms by which these ligands increase the amounts of these AChRs. In particular, we will investigate the hypothesis that nicotine and other membrane permeable cholinergic ligands increase assembly of AChR subunits by acting as molecular chaperones.
Aim 3 is to determine mechanisms which limit the expression of functional ct6 and al AChRs, then to develop cell lines which produce high levels of AChRs to facilitate functional and structural studies of these AChR subtypes. We will investigate the effects of the AChR-associated proteins Ric-3 and lynx-1 on the synthesis of several AChR subtypes and stoichiometries. We will use chimeras and single amino acid mutations to map the sequences which limit proper assembly of alpha6 with beta2 subunits.
Aim 4 is to expand our library of subunit-specific monoclonal antibodies (mAbs). We will use water soluble extracellular domain constructs of human alpha7 AChRs to prepare mAbs to the extracellular domain of native human alpha7 AChRs. We will also prepare mAbs to bacterially-expressed human AChR subunits and native human AChRs purified from our cell lines.
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