Post-translational regulation of neuronal acetylcholine receptors stably expressed in a mouse fibroblast cell line

Rothhut, Bernard; Romano, Suzanne J.; Vijayaraghavan, Sukumar; Berg, Daniela

doi:10.1002/(sici)1097-4695(199601)29:1<115::aid-neu9>3.0.co;2-e

Cited by 31 publications

(24 citation statements)

References 37 publications

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“…It has been suggested that this "persistent inactivation" may be a consequence of the receptor adopting a long-lasting desensitized state. A 2-fold up-regulation in the level of cell-surface ␣4␤2 nAChR has also been reported as a consequence of treatments which elevate intracellular cAMP (26). It has also been shown previously that subunit folding and assembly of some nAChRs, notably those of invertebrates and of cold water fish such as Torpedo, when expressed in mammalian cell lines, is more efficient at lower temperatures (27,28).…”

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confidence: 70%

Up-regulation of Cell-surface α4β2 Neuronal Nicotinic Receptors by Lower Temperature and Expression of Chimeric Subunits

Cooper

Harkness

Baker

et al. 1999

Journal of Biological Chemistry

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The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing ␣4 and ␤2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat ␣4␤2 nAChR. In addition to the relatively well characterized nicotinic acetylcholine receptor (nAChR) 1 expressed at the vertebrate neuromuscular junction, a family of pharmacologically distinct "neuronal" nAChRs is expressed within the central and peripheral nervous system (1, 2). Whereas the muscle-type nAChR is a pentameric complex of known subunit composition (␣ 2 ␤␥␦ in fetal muscle and ␣ 2 ␤⑀␦ in adult), the precise subunit composition of the various neuronal nAChR subtypes is less certain. To date, 11 neuronal nAChR subunits (␣2-␣9 and ␤2-␤4) have been identified and cloned. There is evidence to suggest that the predominant neuronal nAChR subtype expressed in the vertebrate brain contains the ␣4 and ␤2 subunits (3, 4). When co-expressed in Xenopus oocytes, ␣4 and ␤2 co-assemble to form functional nAChRs (5) with a subunit stoichiometry of (␣4) 2 (␤2) 3 (6, 7).Several studies have demonstrated that relatively high levels of functional nAChRs are expressed on the cell surface of mammalian fibroblasts transfected with muscle (␣ 2 ␤␥␦ or ␣ 2 ␤⑀␦) nAChR subunit cDNAs (8, 9). In contrast, it appears that some neuronal nAChR subunit combinations are expressed considerably less efficiently when expressed heterologously in mammalian cell lines. In particular, the neuronal nAChR ␣7 and ␣8 subunits, which readily form functional homo-oligomeric nAChRs when expressed in Xenopus oocytes, appear to fold and assemble very inefficiently in many mammalian cell types (10 -15). In contrast, chimeric subunits containing the extracellular domain of the ␣7 or ␣8 subunits, together with the transmembrane and intracellular regions of the 5HT 3 receptor subunit, produce very high levels of cellsurface expression in all cell types examined (11,12,14,16,17).Functional expression of recombinant ␣4␤2 nAChRs in mammalian cell lines has been demonstrated previously (18 -20), but detailed characterization has been hindered somewhat by relatively low levels of cell-surface expression. Chronic exposure to nicotine has been shown to result in an increase in radioligand binding sites in cell lines expressing recombinant ␣4␤2 nAChRs (21-23), and correlates with an up-regulation (by ϳ2-fold) of the number of cell-surface nAChRs (21). However, despite up-regulation of cell-surface nAChRs, chronic treatment with nicotine has been reported to result in persistent functional inactivation of both recombinant ␣4␤2 and native nAChRs (21,24,25). It has been suggested that this "persistent inactivation" may be a consequence of the receptor adopting a long-lasting desensitized state. A 2-fold up-regulation in the level of cell-surface ␣4␤2 nAChR has also been reported as a consequence of treatments which elevate intracellular cAMP (26). It has also been shown previously ...

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confidence: 70%

Up-regulation of Cell-surface α4β2 Neuronal Nicotinic Receptors by Lower Temperature and Expression of Chimeric Subunits

Cooper

Harkness

Baker

et al. 1999

Journal of Biological Chemistry

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“…The efficiency of subunit assembly can be regulated under certain conditions leading to higher nAChR expression. Studies in mouse fibroblasts showed that increased intracellular cAMP levels increase nAChR assembly efficiency and surface expression [19,20]. Another mechanism emerging as an important modulator of nAChR expression involves association with chaperone proteins that transport receptors away from the endoplasmic reticulum [8].…”

Section: Discussionmentioning

confidence: 99%

Increased expression of α7nAChR in chronic rhinosinusitis: The intranasal cholinergic anti-inflammatory hypothesis

2016

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“…These intracellular nAChRs may consist of two distinct pools: nAChRs destined for immediate surface expression and nAChRs stored as an independent pool. Intracellular nAChR pools in other tissues (Stollberg and Berg, 1987) and recombinant cell lines (Whiteaker et al, 1998) can be modulated (Rothhut et al, 1996) and may be involved with the receptor turnover process. It is also possible that these intracellular nAChRs may represent internalized receptors that were once on the receptor surface.…”

Section: Discussionmentioning

confidence: 99%

“…These include alterations in nAChR formation and assembly (Mitra et al, 2001), nAChR transport to the cell surface (Rothhut et al, 1996;Keller et al, 2001), or nAChR stabilization in the cellular membrane (Peng et al, 1994). The rate-limiting processes involved in nAChR turnover remain to be determined and may vary with different disease conditions.…”

mentioning

confidence: 99%

Expression of Native α3β4* Neuronal Nicotinic Receptors: Binding and Functional Studies Investigating Turnover of Surface and Intracellular Receptor Populations

Free

McKay

Gottlieb³

et al. 2005

Mol Pharmacol

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Several pathological conditions involve alterations in expression of neuronal nicotinic acetylcholine receptors (nAChRs).Although some studies have addressed processes involved with muscle nAChR expression, knowledge of the regulation of neuronal nAChRs is particularly sparse. The following studies were designed to investigate cellular mechanisms involved with expression of neuronal ␣3␤4* nAChRs. Catecholamine secretion assays and receptor binding studies coupled with receptor alkylation were used to study the nAChR regulation and turnover. Alkylation of adrenal nAChRs results in a rapid and complete loss of receptor-mediated neurosecretion and surface [ 3 H]epibatidine binding sites. After alkylation, both neurosecretory function and nAChR binding slowly (24 -48 h) return to prealkylation levels. When cells are treated with the protein synthesis inhibitor puromycin, after alkylation, receptor-mediated neurosecretion does not recover. Long-term treatment (24 -48-h) with puromycin, in the absence of alkylation, results in a slow, time-dependent shift to the right, followed by a downward shift, in the nicotine concentration-response curve, documenting a disappearance of surface nAChRs. Puromycin treatment alone also results in a loss to both surface and intracellular [3 H]epibatidine binding sites. nAChR ␤4 subunit levels are significantly decreased after treatment with puromycin. These data support a constitutive turnover of adrenal ␣3␤4* nAChRs, requiring continual de novo synthesis of new receptor protein.Nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels that have essential physiological roles in the central and peripheral nervous systems and are thought to be the primary mediators of nicotine addiction. Neuronal nAChRs have also been associated with a variety of other neurological disease states, all of which involve changes in expression or distribution of the nAChRs (Lindstrom, 1997). Several processes have been described that regulate neuronal nAChR expression and directly influence their functional activity. These processes have a temporal component. Loss of receptor function via desensitization occurs rapidly, usually within seconds or minutes. Receptor down-regulation develops over several hours. Receptor up-regulation occurs with some neuronal nAChR subtypes and also develops over several hours. Finally, tolerance and dependence generally take days to weeks to develop. Several mechanisms are probably involved with these processes, including alterations in receptor internalization, receptor recycling, receptor degradation, and/or receptor synthesis.At least three separate steps in the receptor expression/ turnover process may regulate the number of nAChRs expressed on the cell surface. These include alterations in nAChR formation and assembly (Mitra et al., 2001), nAChR transport to the cell surface (Rothhut et al., 1996;Keller et al., 2001), or nAChR stabilization in the cellular membrane (Peng et al., 1994). The rate-limiting processes involved in nAChR turnover remain to...

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Post-translational regulation of neuronal acetylcholine receptors stably expressed in a mouse fibroblast cell line

Cited by 31 publications

References 37 publications

Up-regulation of Cell-surface α4β2 Neuronal Nicotinic Receptors by Lower Temperature and Expression of Chimeric Subunits

Up-regulation of Cell-surface α4β2 Neuronal Nicotinic Receptors by Lower Temperature and Expression of Chimeric Subunits

Increased expression of α7nAChR in chronic rhinosinusitis: The intranasal cholinergic anti-inflammatory hypothesis

Expression of Native α3β4* Neuronal Nicotinic Receptors: Binding and Functional Studies Investigating Turnover of Surface and Intracellular Receptor Populations

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