Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

Kirsch, Glenn E.; Fedorov, N. A.; Kuryshev, Yuri A.; Liu, Zhiqi; Armstrong, Lucas C.; Orr, Michael S.

doi:10.1089/adt.2015.688

Cited by 14 publications

(9 citation statements)

References 48 publications

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“…Our laboratory has previously demonstrated the utility of the automated electrophysiology platform, IonWorks Barracuda, for high-throughput analysis of the nAChR subtypes α3β4, α3β4α5, α4β2, and α7. 11 In the present study, we demonstrate the suitability of this high-throughput electrophysiological assay for screening and selectively profiling the human α6/3β2β3 V273S nAChR receptor.…”

Section: Introductionmentioning

confidence: 62%

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High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

et al. 2017

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Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.

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Section: Introductionmentioning

confidence: 62%

“…These cell lines were developed previously in our lab. 11…”

Section: Methodsmentioning

confidence: 99%

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

et al. 2017

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“…Electrophysiological recordings were performed, using standard methods in the field [70, 71], on HEK-293T cell line transfected with full-length cDNAs for the human GABAA receptor subtypes (α1β3γ2, α2β3γ2, α3β3γ2, α4β3γ2 or α5β3γ2). Holding potential was −70mV.…”

Section: Methodsmentioning

confidence: 99%

Symptomatic and Neurotrophic Effects of GABAA Receptor Positive Allosteric Modulation in a Mouse Model of Chronic Stress

Bernardo

Marcotte

et al. 2021

Preprint

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Chronic stress is a major risk factor for developing depressive disorders and animal models of stress recapitulate behavioral, cellular and molecular changes that are observed in human depression. Individuals exposed to chronic stress, or patients with MDD experience mood and cognitive dysfunctions. This is in part due to neuronal shrinkage in brain regions involved in several cognitive functions such as the prefrontal cortex (PFC) and the hippocampus (HPC). Also in the context of depression and chronic stress, expression levels and function of the main inhibitory neurotransmitter GABA are reduced. Thus far, drugs targeting this GABA deficit have failed to produce beneficial effects due to broad activity at various GABA receptor subunits, including the α1-subunit, resulting in broad side effects. However, refined and selective activity at the α2/3/5-subunit is hypothesized to exert beneficial effect, devoid of side effects.Here, we show that GL-II-73 and GL-I-54 exert positive allosteric modulation at the α5, and α2/3/5-contianing GABAA receptors respectively, and that they are effective both independently and in combination. Using unpredictable chronic mild stress (UCMS) experiments in male and female C57BL/6 mice (n=12 per group), we showed that acute and chronic administration of a GL-II-73/GL-I-54 racemic mixture (termed “GL-RM”) reduced anxiety-like phenotypes and reversed a working memory deficit in UCMS exposed mice. Brains from animals receiving chronic treatment were collected and stained using a Golgi staining technique. Using stereological approaches, neuronal morphology was reconstructed and dendritic length, spine count and spine density were assessed in pyramidal neurons of the PFC and hippocampus. Chronic GL-RM rescued spine density depletions caused by UCMS at apical and basal dendrites (PFC and CA1). Interestingly, spine densities in both brain regions were correlated to cognitive performance, confirming ameliorative benefits of GL-RM.Together, results support the value of selectively targeting GABAA receptors, excluding the α1-subunit, to overcome chronic stress-induced mood symptoms and cognitive deficits, as well as detriments in neuronal morphology. This study confirm results that were observed in old mice, using a α5-selective positive allosteric modulator, and reinforce the concept that the α2/3/5-containing GABAA receptor are suitable targets for the treatment of stress-induced disorders.

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“…The nAChR α7 was expressed in Chinese Hamster Ovary (CHO) cell lines as described previously. 24 The CHO cells were maintained in Ham's F-12 media and transfected with the linearized cDNA constructs by nucleofection. The transfected cells were incubated with α7/RIC3 (antibiotics) for 2 weeks to select the stably expressed cells.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Development of a Nicotinic Acetylcholine Receptor nAChR α7 Binding Activity Prediction Model

Sakkiah

Leggett

Pan

et al. 2020

J. Chem. Inf. Model.

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Despite the well-known adverse health effects associated with tobacco use, addiction to nicotine found in tobacco products causes difficulty in quitting among users. Nicotinic acetylcholine receptors (nAChRs) are the physiological targets of nicotine and facilitate addiction to tobacco products. The nAChR-α7 subtype plays an important role in addiction; therefore, predicting the binding activity of tobacco constituents to nAChR-α7 is an important component for assessing addictive potential of tobacco constituents. We developed an α7 binding activity prediction model based on a large training data set of 843 chemicals with human α7 binding activity data extracted from PubChem and ChEMBL. The model was tested using 1215 chemicals with rat α7 binding activity data from the same databases. Based on the competitive docking results, the docking scores were partitioned to the key residues that play important roles in the receptor−ligand binding. A decision forest was used to train the human α7 binding activity prediction model based on the partition of docking scores. Five-fold cross validations were conducted to estimate the performance of the decision forest models. The developed model was used to predict the potential human α7 binding activity for 5275 tobacco constituents. The human α7 binding activity data for 84 of the 5275 tobacco constituents were experimentally measured to confirm and empirically validate the prediction results. The prediction accuracy, sensitivity, and specificity were 64.3, 40.0, and 81.6%, respectively. The developed prediction model of human α7 may be a useful tool for high-throughput screening of potential addictive tobacco constituents.

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Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

Cited by 14 publications

References 48 publications

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

Symptomatic and Neurotrophic Effects of GABAA Receptor Positive Allosteric Modulation in a Mouse Model of Chronic Stress

Development of a Nicotinic Acetylcholine Receptor nAChR α7 Binding Activity Prediction Model

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