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

Abstract

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 α6/3β2β3^V273S 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 α6/3β2β3^V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.

Keywords: automated patch clamp; electrophysiological screening; ion channel; nicotinic acetylcholine receptor.

Figure 1.

Functional expression in IonWorks Barracuda. Cells were thawed from cryogenic storage and dispensed into the assay plate in population patch clamp mode. (A) Current traces in plate view. (B) Sample records at higher magnification. The vertical line indicates application of ligand. The receptors were activated by application of either 0.3 or 3 µM (-)-nicotine. (C) Amplitude distribution. Holding potential, –70 mV. Mean seal resistance ± SD = 422.5 ± 84 MΩ (n = 380 wells). Four wells were invalid (<100 MΩ, shaded wells).

Figure 2.

Agonist assay validation. (A) Concentration-response curves (16-point) were obtained for three full agonists in two independent experiments on different days. Compounds were administered at the indicated concentrations, and responses were recorded simultaneously. (B) Concentration-response curves (eight-point) for partial agonists, varenicline and cytisine. Data points represent mean peak current ± SEM (two to four replicate wells/concentration). Curves were fitted to the data by a one-site model.

Figure 3.

Antagonist mode validation. Concentration-response curves (eight-point) were obtained for reference antagonists in two independent experiments on different days. Cells were preincubated with test compound at the indicated concentrations. Cells were stimulated with (-)-nicotine at a supramaximal concentration (30 µM). Data points represent mean peak current ± SEM (three to four replicate wells/concentration).

Figure 4.

Subtype profiling of known and novel nicotinic acetylcholine receptors inhibitors. Mean IC₅₀ values ± SEM (n = 2–4) obtained in four-point (0.02–20 µM) or eight-point (methyllycaconitine only, 0.001–3 µM) concentration-response curves. The cells were preincubated with the test compounds for 2 min and then stimulated with (-)-nicotine at EC₉₀ concentrations (100 µM for α3β4, α4β2, and α3β4α5; 3 µM for α6/3β2β3^V273S; and 3 µM + 1 µM PNU 120596 for α7).