Nicotinic Acetylcholine Receptors as Targets for Tobacco Cessation Therapeutics: Cutting-Edge Methodologies to Understand Receptor Assembly and Trafficking

Fox-Loe, Ashley M.; Dwoskin, Linda P.; Richards, Christopher I.

doi:10.1007/978-1-4939-3768-4_7

Cited by 5 publications

(4 citation statements)

References 59 publications

(94 reference statements)

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“…GFP constructs were made by inserting the label between the M 3 and M 4 transmembrane segments of the ␣4 subunit. Both constructs have been shown to produce functional receptors in previous studies (25,42,43).…”

Section: Methodsmentioning

confidence: 89%

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Fox-Loe¹,

Moonschi²,

Richards

2017

Journal of Biological Chemistry

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Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure.

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Section: Methodsmentioning

confidence: 89%

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Fox-Loe¹,

Moonschi²,

Richards

2017

Journal of Biological Chemistry

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“…Therefore, to prevent withdrawal symptoms, smokers may need to smoke continuously to maintain plasma nicotine levels and search for environmental contexts that reinforce smoking behavior in order to derive rewarding effects 40 , 41 . Although nicotine activates DA release and has been indicated to be involved in addiction mechanisms, high-dose applications or fully occupied nAChRs may lead to desensitization and a prolonged depression of evoked dopamine release 33 , 42 , 43 .…”

Section: Discussionmentioning

confidence: 99%

Differences in Nicotine Encoding Dopamine Release between the Striatum and Shell Portion of the Nucleus Accumbens

et al. 2018

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Nicotine desensitization is associated with suppression of tonic release of DA in both the striatum and NAc shell that may occur via the α4β2 subtype of nAChR, whereas phasic frequency-dependent augmentation and HFS-related gating release is more pronounced in the striatum than in the NAc shell. Differences between phasic and tonic release associated with nicotine desensitization may underlie processing of reward signals in the NAc shell, and this may have major implications for addictive behavior.

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“…When SEP is fused with plasma membrane resident receptors, the extracellular pH can be manipulated to turn this fluorescence on or off 5,7–9,17 . When the pH of the extracellular solution is a physiological 7.4, receptors from both the plasma membrane and the endoplasmic reticulum within the region of TIRF excitation fluoresce.…”

Section: Respresentative Resultsmentioning

confidence: 99%

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Fox-Loe

Henderson

Richards

2017

JoVE

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SHORT ABSTRACT Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane. LONG ABSTRACT Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

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Nicotinic Acetylcholine Receptors as Targets for Tobacco Cessation Therapeutics: Cutting-Edge Methodologies to Understand Receptor Assembly and Trafficking

Cited by 5 publications

References 59 publications

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Differences in Nicotine Encoding Dopamine Release between the Striatum and Shell Portion of the Nucleus Accumbens

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

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