Hollands EC, Dale TJ, Baxter AW, Meadows HJ, Powell AJ, Clare JJ, Trezise DJ
Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA {alpha}1{beta}3{gamma}2 Channels Expressed in HEK-293 Cells. [JOURNAL ARTICLE]
J Biomol Screen 2009 Jun 25.
gamma-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 alpha1beta3gamma2 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 EC20 response (1 microM). 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 microM) depressed the maximal GABA response, implying a noncompetitive antagonism. Overall, the pharmacology of human alpha1beta3gamma2 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 alpha1beta3gamma2 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.
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