Development and optimization of a high-throughput electrophysiology assay for neuronal α4β2 nicotinic receptors

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Abstract

Historically, the identification of α4β2 nicotinic acetylcholine receptor ligands has been based on high-throughput radioligand binding, rubidium efflux assays and Ca++ flux assays using a fluorometric imaging plate reader (FLIPR). Among other approaches, low-throughput electrophysiological assays in Xenopus oocytes and two channel application “liquid filament” systems for mammalian cells have been commonly used. More recent technical innovations that have been introduced into the field of electrophysiology allow for automated simultaneous multi-channel operation. Here we report the development and optimization of a high-throughput electrophysiological assay for identifying functionally active α4β2 nicotinic receptor ligands using such a system. Characterization of the test system yielded results comparable to those obtained by other investigators using conventional electrophysiological assays. For example, the concentration–response relationships obtained for α4β2 receptor activation by acetylcholine and nicotine were best described by biphasic Hill equations, and the inhibition of α4β2 receptor currents by the nicotinic antagonist dihydro-β-erythroidine was consistent with previously published results. Functional up-regulation of α4β2 receptors by prolonged exposure to nicotine or lower temperature was also confirmed. Using this methodology we were able to characterize the activation of α4β2 receptors by multiple compounds in a mammalian cell expression system, exemplifying its utility for rapid identification of novel nicotinic ligands within a screening cascade. Our results demonstrate the utility of this electrophysiological tool for the discovery of α4β2 nicotinic acetylcholine receptor ligands with potential applications in numerous clinical indications.

Introduction

The high affinity α4β2 receptor subtype, one of the primary neuronal nicotinic acetylcholine receptor subtypes in the brain, has been identified as a key therapeutic target in several neurodegenerative and cognitive disorders such as Alzheimer's disease, attention-deficit/hyperactivity disorder (ADHD) and cognitive dysfunction in schizophrenia. Consequently, in the last two decades the number of α4β2-selective compounds being assessed in clinical trials has increased substantially (Arneric et al., 2007).

Traditionally the identification of nicotinic receptor ligands has been based on high-throughput radioligand binding (Houghtling et al., 1995) and low-throughput functional assays, including rubidium efflux assays (Lukas and Cullen, 1988), electrophysiological assays in Xenopus oocytes (Harvey et al., 1997, Zwart and Vijverberg, 1998) and two channel application “liquid filament” systems for receptors expressed in mammalian cell lines (Buisson and Bertrand, 2001). In recent years, a novel high-throughput assay measuring Ca++ flux with a fluorometric imaging plate reader (FLIPR) was introduced that has proven to be a useful screening tool for identifying nicotinic ligands (Quik et al., 1997, Fitch et al., 2003, Dunlop et al., 2007). However, fluorescence-based high-throughput screening is not able to achieve the precision required to identify compounds that preferentially bind to open, closed or inactivated ion channels. The whole-cell voltage clamp technique, described almost three decades ago (Hamill et al., 1981) has been considered to be the gold standard method for recording ion channel currents carried through ligand- and voltage-gated channels. Patch clamp offers a real time direct measurement of the effects of chemical compounds on ion flow through these channels. It also allows current measurements under conditions where repetitive stimulation (a phenomenon that naturally occurs in excitable tissues) is applied to the test system.

Although informative and precise, traditional electrophysiological methods are labor and cost intensive, require a skilled investigator and generate very few data points per experimental day. Several years ago, the Dynaflow® system was introduced by Cellectricon (Sinclair et al., 2003, Olofsson et al., 2004). This system still required all of the components of a manual patch clamp setup, but included a fast solution exchange feature using up to 48 channels, with low throughput capacity. More recent technical innovations that have been introduced into the field of electrophysiology include the PatchXpress® 7000A (Tao et al., 2004) and QPatch (Mathes, 2006). Both devices are medium-throughput fully automated multi-channel systems that allow simultaneous recording from 16 (PatchXpress® 7000A) or 48 (QPatch) channels. Although both platforms have been used extensively for studying voltage-gated ion channels (Dubin et al., 2005, Guo and Guthrie, 2005, Dunlop et al., 2007, Trepakova et al., 2007, Korsgaard et al., 2009), there are few reports of their implementation in screening cascades for ligand-gated channels (Dunlop et al., 2007, Friis et al., 2009). Here we describe the development and optimization of a high-throughput assay for screening and identifying α4β2 neuronal nicotinic receptor-selective ligands using a PatchXpress® 7000A parallel patch–clamp system.

Section snippets

Cell culture and harvesting

The human epithelial SH-EP1 cell line stably expressing α4β2 neuronal nicotinic receptors was obtained from Dr. Ronald J. Lukas (Barrow Neurological Institute, Phoenix, AZ). Cells were routinely grown at 37 °C under a 95% O2/5% CO2 atmosphere, in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) and 130 ng/mL Geneticin (MediaTech, Manassas, VA). For low-temperature up-regulation studies plates were

Acetylcholine concentration–response

Typical electrophysiological responses in SH-EP1 cells stably transfected with human α4β2 receptors are shown in Fig. 1A. The best fit of the concentration–response curve was obtained using two Hill equations (Fig. 1B, solid line), consistent with the presence of two known stoichiometries of the α4β2 nicotinic receptor subtype, one having high sensitivity ([α4]2[β2]3) and the other low sensitivity ([α4]3[β2]2) to ligand activation (Moroni et al., 2006, Briggs et al., 2006). EC50 values for the

Discussion

Here we report a detailed electrophysiological characterization of a neuronal nicotinic receptor subtype using an automated parallel patch clamp system. Our data provide proof of principle that such a system can be successfully integrated into a screening cascade for ligand-gated ion channels and particularly for nicotinic receptors. To date the system is typically capable of generating 300–400 data points per day which translates into the evaluation of 20–30 compounds/day.

In SH-EP1 cells stably

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