PrecisION hGABA_A α1/β3/γ2-HEK Recombinant Cell Line

PrecisION hGABA_A α1/β3/γ2-HEK Recombinant Cell Line

PrecisION

2 mL

Recombinant HEK293 cell line expressing the human GABA_A α1/β3/γ2 chloride ion channel

GABA is the major inhibitory neurotransmitter in the CNS, binding to fast-acting ionotropic GABA_A receptors to cause inward flux of Cl- resulting in membrane hyperpolarization and thus reducing membrane excitability (Nutt et al., 2006). Excessive stimulation of these receptors can lead to sedation and ataxia whereas attenuation leads to arousal, insomnia and anxiety. Since modulation of these receptors has such profound physiological effects they have become important drug targets for the treatment of many conditions e.g. anxiety, epilepsy, sleep disorders and for anesthesia (Rudolph and Mohler, 2006). GABA_A receptors are pentameric structures typically consisting of α, β and γ2 subunits in a stoichiometry of 2:2:1. The specific subunit composition is especially important since various combinations mediate different effects (Rudolph and Mohler, 2006). For example, α1-containing receptors, accounting for 60% of all GABA_A receptors, mediate the sedative/hypnotic effects of benzodiazepines (BZPs) whereas the anxiolytic effects of these drugs are mediated by receptors containing α2 and α3 subunits (Korpi and Sinkkonen, 2006). Hence, developing selective allosteric modulators for these latter subunits should ultimately lead to anxiolytic drugs devoid of unwanted sedative effects.

HEK293 cells expressing human GABA_A 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 µM (mean, n=16). Bicucculine, a known GABA_A antagonist, abolished (>98% block at 11 µM) responses to 10 µM GABA. This antagonism was demonstrated to be competitive, with a pA2 value of 6.1. The GABA blocker picrotoxin produced inhibition of the 30 µM GABA response with a mean IC50 value of 3.2 µM. The benzodiazepines pentobarbital, tracazolate and zolpidem all positively modulated the 1 µM 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 hGABA_A α1/β3/γ2 currents >500 pA with a mean current amplitude of 2.76 nA ± 0.21 nA (n=183).

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel.

Human

Mycoplasma Testing:
The cell line has been screened to confirm the absence of Mycoplasma species

HEK

Ion Channel Cell Lines

Pack contains 2 vials of mycoplasma-free cells, 1 ml per vial

Upstate

293 cell line

2 x 1 ml aliquots

hGABA_A α1/β3/γ2

Chloride

GABRA1

Ion Channels

PrecisION hGABA_A α1/β3/γ2-HEK Recombinant Cell Line