Pharmacology of nicotinic acetylcholine receptor (nAChR) upregulation in the transfected cell line, M10

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 ␣3␤2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for ␣3␤4 receptors, offering a rationale to investigate the molecular mechanism underlying upregulation. 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 ␤2/␤4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two ␤2 segments, 74 -89 and 106 -115, confer up-regulation to ␤4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the ␣3␤2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The ␤4 microdomain is sufficient to confer to ␤2 a ␤4-like upregulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between ␣4␤2 and ␣4␤4. 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 ␣4␤2 is the subtype exhibiting the highest degree of up-regulation in the brain.Nicotine is the primary substance responsible for tobacco addiction, a major cause of death in western societies (1, 2). Chronic exposure to nicotine causes a strong addiction, which is mediated by the interaction of nicotine with neuronal nicotinic acetylcholine receptors (nAChRs), 1 a class of pentameric allosteric ligand-gated ion channels engaged in cholinergic nicotinic transmission in the brain (3, 4).Post-mortem analysis of brain slices from smokers (5, 6) and from rats or mice chronically treated with nicotine (7-10) reveals large increases in the number of high affinity nicotinic binding sites, a phenomenon termed up-regulation. Nicotine up-regulates the ␣3␤2 (11, 12), ␣4␤2 (13-18), ␣7 (11, 19), and (␣1)2␤1␥␦ (19) nAChR subtypes reconstituted in cell lines or in Xenopus oocytes.In addition to the increased number of sites, modulation of the magnitude of the nicotine-elicited electrophysiological response is also observed upon chronic nicotine incubation of cell lines expressing recombinant nAChRs. This functional potentiation of the response is observed with nAChR oligomers ␣4␤2 and ␣3␤2 reconstituted in mammalian cell lines (12,20). But functional depression almost systematically takes place with the same combination of subunits reconstituted in Xenopus oocytes...

Section: Different Contributions Of the ␣ And ␤ Subunits To The Upregsupporting

confidence: 67%

Section: Up-regulation Involves An Increase In Intracellular Bindingmentioning

confidence: 60%

Section: Different Contributions Of the ␣ And ␤ Subunits To The Upregmentioning

confidence: 99%

Section: Different Contributions Of the ␣ And ␤ Subunits To The Upregmentioning

confidence: 99%

Section: Construction Of Chimeras Between ␤2mentioning

confidence: 99%

See 3 more Smart Citations

An Extracellular Protein Microdomain Controls Up-regulation of Neuronal Nicotinic Acetylcholine Receptors by Nicotine

Sallette

Bohler

Benoit

et al. 2004

Agonist-dependent Up-regulation of Human Somatostatin Receptor Type 1 Requires Molecular Signals in the Cytoplasmic C-tail

Hukovic¹,

Rocheville²,

Kumar

et al. 1999

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 M somatostatin (SST)-14 ؋ 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (⌬) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, ⌬ C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.

Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Hussmann

Yasuda

Xiao

et al. 2011

Background: Nicotinic ligands up-regulate neuronal nicotinic receptors in vitro and in vivo. Results: Carbachol or oxotremorine plus nicotine up-regulate ␣4␤2 nicotinic receptors more than nicotine alone. Conclusion: Muscarinic receptor activity augments nicotinic receptor up-regulation by increasing ␣4 and ␤2 subunit mRNA and protein.Significance: This study reveals a novel target for modulating neuronal nicotinic receptor expression.