Nicotine upregulates its own receptors through enhanced intracellular maturation.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smoker's brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

Regional differential effects of chronic nicotine on brain alpha 4-containing and alpha 6-containing receptors.

Chronic nicotine upregulates central nicotinic acetylcholine receptors (nAChRs), a plasticity process thought to contribute to its addictive properties. To analyze this process in vivo, we chronically exposed mice to nicotine using minipump delivering nicotine at concentrations close to those found in tobacco smokers. Binding studies show upregulation of high-affinity nAChRs after 21 days of treatment in cortical areas, caudate putamen, nucleus accumbens, hippocampus, ventral tegmental area, and superior colliculi. No upregulation was observed in thalamus and discrete cortical areas. Using wild type and alpha 6-/- mice, we observed a downregulation of alpha 6*-nAChRs in superior colliculi and no effects in other structures. The complex pattern of upregulation/downregulation observed in this study depends on both nAChR composition and regional distribution.

Distinct subcellular targeting of fluorescent nicotinic alpha 3 beta 4 and serotoninergic 5-HT3A receptors in hippocampal neurons.

The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of alpha3beta4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric alpha3CFP-beta4 and YFP-alpha3beta4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, alpha3beta4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of alpha3beta4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.

An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine.

In smoker's brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.