Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Abstract

γ-Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC 20 response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA2 and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human α1β3γ2 GABAA determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z′ values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the α1β3γ2 GABAA isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABAA receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

Validation and Optimization of Novel High-Throughput Assays for Human Epithelial Sodium Channels

“…[22][23][24][25][26] Cells were prepared immediately prior to experimentation as previously described 26 and resuspended in warm external buffer (in mM, Na-gluconate 120, KCl 5, NaCl 20, CaCl 2 1, MgCl 2 2, HEPES 10, pH adjusted to 7.35 with NaOH, 0.2 µm filter sterilized) to yield a final cell density of ~2 × 10 6 cells per mL. Internal buffer was composed of (mM) K-gluconate 120, NaCl 5, KCl 20, HEPES 10, CaCl 2 2, MgCl 2 1, pH adjusted to 7.2 with KOH (0.2 µm filter sterilized).…”

“…26 A holding potential (V m ) of −40 mV was applied for 5 s followed by a ramp (250 ms, −100 mV to +100 mV with a 20 ms hold at each extremity) and step (50 ms, 0 mV; 50 ms, +30 mV) protocol. The corresponding currents were recorded first in the absence (pre-) and then the presence (post-) of amiloride, test compound, or vehicle (compound incubation time of 3-5 min).…”

“…Taking learnings from assays for other non-voltage-gated channels, 24,26 we next developed a planar array patch clamp electrophysiological assay for ENaC using IonWorks Quattro. In single-hole experiments on control HEK293 β*γ*-expressing cells, grown in standard media without the α-subunit, the measured seal resistance was 197.1 ± 63.6 MΩ (n = 366).…”

Determining the Relative Efficacy of Positive Allosteric Modulators of the GABAA Receptor: Design of a Screening Approach

“…Indeed, the cellular response to Gaba (e.g., Gaba at EC 20 ) is dependent on GABA A receptor expression levels and receptor combinations (different subtypes or αβ vs. αβγ containing receptors), and such heterogeneity may be expected during expression of recombinant GABA A receptors. 13 In this study, we selected zolpidem as an internal comparator because of its medium GABA A affinity and its convenient washout properties. We evaluated the activity of zolpidem on the human α1β2γ2S GABA A receptor stably expressed in a CHO cell line by using the automated patch clamp system.…”

“…Different methods have been described that allow for the functional screening of GABA A receptors including 36 Cl -flux, 8 I -influx, 9,10 fluorescence resonance energy transfer assay, 11 FLIPR assay, 12 and automated patch clamp. 13 Although these screening methods have been successfully used to identify and to determine GABA A modulator potencies, only a few have so far studied the accurate measurement of the relative efficacy of the GABA A modulators. Analyzing the relative efficacy of GABA A PAMs requires appropriate experimental protocols in which agonist concentration, reference compound, and GABA A receptor expression are monitored.…”