Population Patch-Clamp Electrophysiology Analysis of Recombinant GABA A 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 GABA_A 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 GABA_A receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABA_A channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The I_GABA was linear and reversed close to the theoretical E_Cl (—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 GABA_A 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 GABA_A isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABA_A receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)