Population patch-clamp electrophysiology analysis of recombinant GABAA alpha1beta3gamma2 channels expressed in HEK-293 cells.
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 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 alpha1beta3gamma2 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 pEC(50) 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 microM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA(2) 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 non-competitive antagonism. Overall, the pharmacology of human alpha1beta3gamma2 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 alpha1beta3gamma2 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.
Hollands
Emma C
EC
Biochemical & Cellular Targets, Molecular Discovery Research, GlaxoSmithKline R&D, Harlow, Essex, United Kingdom.
Dale
Tim J
TJ
Baxter
Andrew W
AW
Meadows
Helen J
HJ
Powell
Andrew J
AJ
Clare
Jeff J
JJ
Trezise
Derek J
DJ
D001569:Benzodiazepines / Q000494:pharmacology; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D055724:Electrophysiological Phenomena / Q000187:drug effects; D005442:Flumazenil / Q000494:pharmacology; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D018408:Patch-Clamp Techniques / Q000379:methods*; D021122:Protein Subunits / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D005442:Flumazenil