MOLECULAR MECHANISMS OF NICOTINE DEPENDENCE

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As a model for the molecular basis of drug addiction and abuse, the proposed studies will establish mechanisms by which chronic nicotine exposure regulates expression and function of diverse nicotinic acetylcholine receptor (nAChR) subtypes. Different nAChR subtypes are expressed by normal and transformed muscle cells or by different neurons and their clonal line analogs. For each combination of normal or clonal cell type and nAChR subtype, chronic nicotine treatment has distinctive effects of nAChR expression. The central hypothesis of this proposal is that effects of nicotine on nAChR expression in brain and in selected cell lines are mediated at both transcriptional and post-translational levels, whereas effects on nAChR in muscle, autonomic ganglia, and analogous cell lines are mediated primarily post-translationally. We hypothesize that, for all cell types, nicotine interaction with open channel blocking sites on nAChR induces an initial, rapid loss of cell surface nAChR and nAChR functional activity. In addition, for brain neurons and for selected cell lines, we hypothesize that there is a subsequent activation of nAChR gene transcription and the reappearance of cell surface nAChR that are functionally silent, at least initially. We also postulate that these functionally silent nAChR have either unique subunit compositions or phosphorylation states and/or need to undergo a maturation process before they exhibit functional activity, and that internalization of nAChR (and/or a change in nAChR phosphorylation state) is the mechanism for early nicotine effects. The proposed study will test these hypotheses by using clonal cell lines that stably and naturally express different nAChR subtypes and either neuronal or muscle characteristics. Time- and dose-dependent effects of treatment with nicotine, and with other nicotinic drugs, will be quantified with respect to (a) levels of nAChR functional activity (assessed by ion efflux assays and single channel recording), (b) levels and sites of nAChR expression (assessed by ligand or antibody binding assays and subcellular distribution analysis), (c) subunit composition of nAChR (assessed by Western immunoblot and affinity isolation analyses), (d) levels of nAChR subunit mRNA expression and gene transcriptional activity (measured using Northern and nuclear run-on assays), and, if warranted, (e) phosphorylation state of nAChR peptides (assessed by 32P labeling in situ and anti- phosphorylation state of nAChR peptides (assessed by 32P labeling in situ and anti-phosphotyrosine immunoblot analyses).