详细信息:Recombinant HEK293 cell line expressing the human GABAA α1/β3/γ2 chloride ion channel. HEK293 cells expressing human GABAA α1, β3 and γ2 subunits were characterised in terms of their pharmacological and biophysical properties using manual patch clamp techniques (perforated patch clamp and conventional whole cell recording) and automated planar array electrophysiology (IonWorks™ Quattro). Using manual patch clamp techniques, 1 mM GABA produced a mean peak current amplitude of 2.21 ± 0.28 nA (mean ± SEM, n=10). The mean current density was 176.5 ± 26.5 pA/pF (mean ± SEM, n=10). On IonWorks™ Quattro the GABA concentration response relationship yielded an EC50 value of 1.96 ™M (mean, n=16). Bicucculine, a known GABAA antagonist, abolished (>98% block at 11 ™M) responses to 10 ™M GABA. This antagonism was demonstrated to be competitive, with a pA2 value of 6.1. The GABA blocker picrotoxin produced inhibition of the 30 ™M GABA response with a mean IC50 value of 3.2 ™M. The benzodiazepines pentobarbital, tracazolate and zolpidem all positively modulated the 1 ™M GABA response, by at least 300%, confirming the presence of the γ2 subunit. Using manual patch clamp techniques, measurement of the zolpidem potentiation of GABA response showed this subunit expression to be stable over time. Functional channel expression over time was monitored using IonWorks™ HT. Channel expression is robust over at least 32 passages. At passage 32, 83% of cells sealed with a resistance >50 MOhm. Of these, 94% expressed hGABAA α1/β3/γ2 currents >500 pA with a mean current amplitude of 2.76 nA ± 0.21 nA (n=183).
