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High-throughput patch clamp screening in human α6-containing nicotinic acetylcholine receptors

In this article, we provide a summary of recent research by Charles River Laboratories, published in SLAS Discovery.

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Feb 22, 2018

A research team at Charles River Laboratories (MA, USA), has described the development and validation of a recombinant cell line expressing α6/3β2β3V273S nicotinic acetylcholine receptors (nAChRs) to be used in screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda, Molecular Devices; CA, USA). The study suggests high-throughput patch clamp screening is a valuable method for the evaluation of nicotinic subtype selectivity and could be utilized to advance tobacco regulatory science by providing a screening method that identifies tobacco product constituents acting at the α6β2β3 nicotinic receptors.

Specific nAChRs are characterized by their α- and β-subunit composition and it is this that determines the pharmacological and biophysical properties of the receptor. Subunits containing α6 have been found primarily in the retina and catecholaminergic nuclei, and α6β2β3 in particular is expressed in the terminal regions of dopaminergic neurons that project to the nucleus accumbens and striatum. Receptors containing α6 are known to modulate dopamine release in the regions of the brain associated with nicotine addiction.

Receptors containing α6 are difficult to investigate due to poor in vitro performance in heterologous expression systems. This has been overcome by creating chimeras with improved functional expressions in cells. The study demonstrated that α6/3β2β3V273S mimicked the function of native α6β2β3 nicotinic receptors, in regards to the ligand-binding site and the suitability of the high-throughput electrophysiology platform for analysis of the nAChR subtype, α6/3β2β3V273S.

The α6/3β2β3V273S nAChR was expressed in human embryonic kidney (HEK)-293 cells, demonstrated by the study to exhibit appropriate functional and molecular characteristics to enable high-throughput automated assays. When analyzed electrophysiologically, the α6/3β2β3V273S-HEK cell line exhibited the expected response to nicotine and showed promise as a test system for pharmacological assays to quantify sensitivity of the AChR to both agonists and antagonists.

The α6/3β2β3V273S-HEK cell line was validated by demonstrating the capability of the test system to reproduce reference compound pharmacology previously reported in literature. As expected, the receptor showed high sensitivity to the agonists nicotine, epibatidine and acetylcholine, and varenicline and cytisine behaved as partial agonists. Antagonist validation was also carried out, including mecamylamine, dihydro-β-ertyhroidine, bupropion, SR 16584 and methyllycaconitine. These results were also consistent with previous analyses by manual patch clamp and fluorescence assays.

The capability of the assay to identify subtype-selective inhibitors was demonstrated by pilot library screening and profiling. The group evaluated the effects of 786 FDA-registered drugs to test the ability of the system to identify agonists and antagonists of the AChR. The study suggests high-throughput automated patch clamp could be a potentially valuable method to analyze the selectivity of inhibitors to AChR sub-types and provides a rapid screening method to identify molecules that act on the α6β2β3 nicotinic receptors.

Read the full research article here

Armstrong LC, Kirsch GE, Fedorov NB, et al. High-throughput patch clamp screening in human α6-containing nicotinic acetylcholine receptors. SLAS Discov. doi:10.1177/2472555217696794 (2017)

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