Nicotine is the major physiologically active component of tobacco that is responsible for the establishment and maintenance of chronic tobacco use. Nicotine exerts its actions through interactions with a diverse population of nicotinic acetylcholine receptors (nAChRs), composed of pentameric assemblies of homologous subunits. Different subunit combinations produce different nAChR subtypes, with diverse properties and distributions. Because of their central role in mediating nicotine's effects, knowing which nicotinic subtypes are expressed, their distribution, and their characteristics is vitally important to understanding the initiation and maintenance of tobacco use. The central hypothesis directing the proposed studies is that established techniques for probing nAChRs, while powerful, are inherently limited by their inability to positively isolate and identify nAChR subtypes on the basis of their component subunits. Immunochemical techniques address this limitation, giving them the potential to greatly expand our knowledge of nAChR composition and properties.
2 specific aims are proposed: 1. Develop immunoprecipitation/immunocapture protocols for neuronal nAChRs. 2. Establish Western blotting techniques for neuronal nAChRs. We have assembled an extensive collection of antibody stocks, directed against the full range of mammalian neuronal nAChR subunits expressed. The performance of each antibody stock in immunoprecipitation/immunocapture and Western blotting techniques will be assessed and optimized. 2 major difficulties are associated with the use of antibodies: they may fail to recognize the target (due to poor affinity or unfavorable experimental conditions), or crossreactions may occur. nAChR subunit-null mutant animals will be used as negative controls to assess the severity of both of these problems. Since null-mutant tissue should express largely the same complement of antigens as found in wild-type tissue, with the exception of the targeted gene product, we anticipate that the null mutants will excel in this capacity.
Aim 1 will be pursued first, since each subunit-specific protocol established may be used immediately to separate and analyze the composition of native nAChRs. This approach has already produced novel data (see Preliminary Results), and further quick scientific returns are anticipated for this approach. [125l]Epibatidine binding techniques developed in our laboratory will be used to monitor the separation of nAChR subtypes, and to determine the pharmacology of those captured. Work on Aim 2 will begin in the second year, since it is likely that these experiments will require more intensive optimization than those covered in Aim 1 and, in any case, Western blotting techniques will be facilitated by the availiblity of nAChR sources purified by immunoprecipitation.
