Development and optimization of a high-throughput electrophysiology assay for neuronal α4β2 nicotinic receptors
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
References (37)
- et al.
Neuronal nicotinic receptors: a perspective on two decades of drug discovery research
Biochem Pharmacol
(2007) - et al.
Ligand-gated ion channels in acutely dissociated rat hippocampal neurons with long dendrites
Neurosci Lett
(1996) - et al.
Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits
J Biol Chem
(1999) - et al.
Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp
J Biomol Screen
(2005) - et al.
In vitro screening strategies for nicotinic receptor ligands
Biochem Pharmacol
(2007) - et al.
Characterization of compounds on nicotinic acetylcholine receptor alpha7 channels using higher throughput electrophysiology
J Neurosci Methods
(2009) - et al.
Automated electrophysiology in the preclinical evaluation of drugs for potential QT prolongation
J Pharmacol Toxicol Methods
(2005) - et al.
Rat nicotinic acetylcholine receptor alpha2beta2 channels: comparison of functional properties with alpha4beta2 channels in Xenopus oocytes
Neuroscience
(2004) - et al.
Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern
J Pharmacol Toxicol Methods
(2004) - et al.
An isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines
Anal Biochem
(1988)
beta-Amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells
J Biol Chem
Nicotinic acetylcholine sensitivity of rat petrosal sensory neurons in dissociated cell culture
Brain Res
Nicotinic receptor function in the mammalian central nervous system
Ann N Y Acad Sci
Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes
J Pharmacol Exp Ther
High- and low-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs
J Mol Neurosci
Chronic exposure to nicotine upregulates the human (alpha)4((beta)2 nicotinic acetylcholine receptor function
J Neurosci
Human alpha4beta2 neuronal nicotinic acetylcholine receptor in HEK 293 cells: a patch–clamp study
J Neurosci
The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperature
J Physiol
Cited by (7)
A method for bidirectional solution exchange-" Liquid bullet" applications of acetylcholine to α7 nicotinic receptors
2012, Journal of Neuroscience MethodsCitation Excerpt :The fastest known solution exchange rate achieved is around 100 μs with θ-electrodes mounted on a piezoelectric translator using excised membrane patches. These methods provide a significantly faster solution exchange rate (Franke et al., 1987) as compared to classical oocytes (Papke and Thinschmidt, 1998; Papke and Porter Papke, 2002) and cell based techniques (Sidach et al., 2009). Several potential disadvantages however must be considered when applying the techniques of fast solution exchange to whole-cell configurations of recording.
Shifting the paradigm: new approaches for characterizing and classifying neurons
2009, Current Opinion in NeurobiologyCitation Excerpt :Additional data resources have been or are being generated for mouse spinal cord, developing mouse brain, rhesus macaque and adult human brain. Analogous to the scaling of histological methods described above, effort is being put into developing methods for high-throughput electrophysiology (http://bluebrain.epfl.ch/) [7,8] and quantitative analysis of cellular morphology, the latter being the topic of a new scientific competition called the DIADEM Challenge (http://diademchallenge.org/) designed to motivate development of computer algorithms to improve the automated reconstruction of neuronal arbors in labeled neurons. This push to scale up traditional neuroscience techniques has also recently engulfed connectivity studies, with various strategies put forward for high-throughput approaches to large-scale connectivity mapping [9,10].
Discovery of peptide ligands through docking and virtual screening at nicotinic acetylcholine receptor homology models
2017, Proceedings of the National Academy of Sciences of the United States of AmericaPhysical and virtual screening methods for marine toxins and drug discovery targeting nicotinic acetylcholine receptors
2013, Expert Opinion on Drug DiscoveryValidation of a high-throughput, automated electrophysiology platform for the screening of nicotinic agonists and antagonists
2013, Journal of Biomolecular Screening