Cellular trafficking of nicotinic acetylcholine receptors

John, Paul A. St.

doi:10.1038/aps.2009.76

Cited by 32 publications

(27 citation statements)

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“…Studies have shown that nAChRs change their cellular expression in response to chronic nicotine exposure in culture and in vivo 28–30 . In order to test the functionality of the epitope-tagged receptor clones and to measure nAChR plasma membrane expression we performed on-cell ELISA with non-permeabilized cells after overnight exposure of the cells to vehicle or 1 mM nicotine (fig.…”

Section: Resultsmentioning

confidence: 99%

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

Ray¹,

Soderblom²,

Bai³

et al. 2017

ACS Chem. Biol.

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Nicotinic acetylcholine receptors regulate the nicotine dependence encountered with cigarette smoking, and this has stimulated a search for drugs binding the responsible receptor subtypes. Studies link a gene cluster encoding for α3β4α5-D398N nicotinic acetylcholine receptors to lung cancer risk as well as link a second mutation in this cluster to an increased risk for nicotine dependence. However, there are currently no recognized drugs for discriminating α3β4α5 signaling. In this study we describe the development of homogenous HEK-293 cell clones of α3β4 and α3β4α5 receptors appropriate for drug screening and characterizing biochemical and pharmacological properties of incorporated α5 subunits. Clones were assessed for plasma membrane expression of the individual receptor subunits by mass spectrometry and immunochemistry and their calcium signaling was measured in the presence of a library of kinase inhibitors and a focused library of acetylcholine receptor ligands. We demonstrated an incorporation of two α3 subunits in approximately 98% of plasma membrane receptor pentamers indicating a 2/3 subunit expression ratio of α3 to β4 alone or to coexpressed β4 and α5. With prolonged nicotine exposure the plasma membrane expression of receptors with and without incorporated α5 increased. Whereas α5 subunit expression decreased the cell calcium response to nicotine and reduced plasma membrane receptor number, it partially protected receptors from nicotine mediated desensitization. Hit compounds from both libraries suggest the α5 and α5-D398N subunits allosterically modify the behavior of nicotine at the parent α3β4 nicotinic acetylcholine receptor. These studies identify pharmacological tools from two distinct classes of drugs, antagonists and modifiers that are α5 and α5-D398N subtype selective that provide a means to characterize the role of the CHRNA5/A3/B4 gene cluster in smoking and cancer.

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Section: Resultsmentioning

confidence: 99%

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

Ray¹,

Soderblom²,

Bai³

et al. 2017

ACS Chem. Biol.

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“…Detailed measures of smoking status such as carbon monoxide, nicotine, or cotinine are needed to assess their effects on [ 18 F]nifene binding. Lastly, recent studies have reported on cellular trafficking of nAChR (reviewed in St John, ). Since [ 18 F]nifene exhibited relatively fast kinetics which is partially attributed to the lower basicity of the 3‐pyrrolinyl ring, our expectation is that it may not be sequestered in intracellular vesicles.…”

Section: Discussionmentioning

confidence: 99%

[¹⁸F]Nifene test-retest reproducibility in first-in-human imaging of α4β2* nicotinic acetylcholine receptors

Lao

Betthauser

Tudorascu

et al. 2017

Synapse

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The aim of this study was to examine the suitability of [18 F]nifene, a novel α4β2* nicotinic acetylcholine receptor (nAChR) radiotracer, for in vivo brain imaging in a first-in-human study. Methods Eight healthy subjects (4 M,4 F;21–69,44 ±21 yrs) underwent a [18F]nifene positron emission tomography scan (200 ±3.7 MBq), and seven underwent a second scan within 58 ±31 days. Regional estimates of DVR were measured using the multilinear reference tissue model (MRTM2) with the corpus callosum as reference region. DVR reproducibility was evaluated with test–retest variability (TRV) and intraclass correlation coefficient (ICC). Results The DVR ranged from 1.3 to 2.5 across brain regions with a TRV of 0–7%, and did not demonstrate a systematic difference between test and retest. The ICCs ranged from 0.2 to 0.9. DVR estimates were stable after 40 min. Conclusion The binding profile and tracer kinetics of [18F]nifene make it a promising α4β2* nAChR radiotracer for scientific research in humans, with reliable DVR test–retest reproducibility.

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“…Thus, RIC-3 has an additional role of transporting the receptor to the surface. In fact, since proper assembly is a pre-requisite for exit of the receptor from the ER [42] the sole ability of RIC-3 to enhance receptor assembly could promote its release from the ER and procession along the secretory pathway.…”

Section: Discussionmentioning

confidence: 99%

RIC-3 differentially modulates α4β2 and α7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation

et al. 2013

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BackgroundRecent work has shown that the chaperone resistant to inhibitors of acetylcholinesterase (RIC-3) is critical for the folding, maturation and functional expression of a variety of neuronal nicotinic acetylcholine receptors. α7 nicotinic receptors can only assemble and functionally express in select lines of cells, provided that RIC-3 is present. In contrast, α4β2 nicotinic receptors can functionally express in many cell lines even without the presence of RIC-3. Depending on the cell line, RIC-3 has differential effects on α4β2 receptor function – enhancement in mammalian cells but inhibition in Xenopus oocytes. Other differences between the two receptor types include nicotine-induced upregulation. When expressed in cell lines, α4β2 receptors readily and robustly upregulate with chronic nicotine exposure. However, α7 nicotinic receptors appear more resistant and require higher concentrations of nicotine to induce upregulation. Could the coexpression of RIC-3 modulate the extent of nicotine-induced upregulation not only for α7 receptors but also α4β2 receptors? We compared and contrasted the effects of RIC-3 on assembly, trafficking, protein expression and nicotine-induced upregulation on both α7 and α4β2 receptors using fluorescent protein tagged nicotinic receptors and Förster resonance energy transfer (FRET) microscopy imaging.ResultsRIC-3 increases assembly and cell surface trafficking of α7 receptors but does not alter α7 protein expression in transfected HEK293T cells. In contrast, RIC-3 does not affect assembly of α4β2 receptors but increases α4 and β2 subunit protein expression. Acute nicotine (30 min exposure) was sufficient to upregulate FRET between α4 and β2 subunits. Surprisingly, when RIC-3 was coexpressed with α4β2 receptors nicotine-induced upregulation was prevented. α7 receptors did not upregulate with acute nicotine in the presence or absence of RIC-3.ConclusionsThese results provide interesting novel data that RIC-3 differentially regulates assembly and expression of different nicotinic receptor subunits. These results also show that nicotine-mediated upregulation of α4β2 receptors can be dynamically regulated by the presence of the chaperone, RIC-3. This could explain a novel mechanism why high affinity α4β2 receptors are upregulated in specific neuronal subtypes in the brain and not others.

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Cellular trafficking of nicotinic acetylcholine receptors

Cited by 32 publications

References 55 publications

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

[¹⁸F]Nifene test-retest reproducibility in first-in-human imaging of α4β2* nicotinic acetylcholine receptors

RIC-3 differentially modulates α4β2 and α7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation

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Cellular trafficking of nicotinic acetylcholine receptors

Cited by 32 publications

References 55 publications

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

[18F]Nifene test-retest reproducibility in first-in-human imaging of α4β2* nicotinic acetylcholine receptors

RIC-3 differentially modulates α4β2 and α7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation

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[¹⁸F]Nifene test-retest reproducibility in first-in-human imaging of α4β2* nicotinic acetylcholine receptors