• GABAA a5b3g2

    Pharmacology on GABAA α5β3γ2 as recorded on the SyncroPatch 96, a predecessor model of the SyncroPatch 384PE

GABAA Receptor Alpha 5 Beta 3 Gamma 2

Family:
GABA Receptors (Cys-loop ligand-gated ion channels)

Subgroups:
The GABA receptor family has further been divided into GABAA, GABAB and GABAA-ρ (also called GABAC) receptors.

Topology:
As typical for Cys-loop receptors, generally four transmembrane spanning domains TM1-TM4 form one receptor subunit, five or four subunits form a pore, several heteromeric combinations are known.
GABAA: Oligomerization of typically five subunits, heteromeric combinations are known of α subunits (GABRA1 - GABRA6), β subunits (GABRB1 - GABRB3) and γ subunits (GABRG1 - GABRG3), as well as δ (GABRD), an ε (GABRE), a π (GABRP), and a θ (GABRQ). The most common type is (α1)2(β2)2(γ2), minimal requirement is  the inclusion of α and β subunits.
GABAB: Heterodimer (GABBR1, GABBR2)
GABAA: Oligomerization of five subunits, heteromeric combinations of three GABAA-ρ subunits are known (GABRR1, GABRR2, GABRR3)

Regulation and Function:
GABAA and GABAA-ρ receptors are ligand-gated ion channels (ionotropic receptors); whereas GABAB receptors are G protein-coupled receptors (metabotropic receptors). All respond to the neurotransmitter gamma-aminobutyric acid (GABA), the main inhibitory transmitter in the mammalian CNS.

Data and Applications

GABAA Receptor (a5b3g2) - Success Rates

Bar Seal cm Rs GABAa5AppNote

icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) data and applications:
Cells were kindly provided by Millipore.

Statistic of hGABAA α5β3γ2 cells recorded on one NPC-96 patch clamp chip. Cslow = 26.2 ±1.8 (n=32), Rs = 4.1 ± 0.2 (n=32). 53 % of the cells on one NPC-96 chip (total n=96) had seal resistance > 1 Giga Ohm at the beginning, 47 % at the end of experiment. 76 % of cells reached a seal resistance above 500 MΩ, which remained constant throughout the experiment.

 

 

 

GABAA Receptor (a5b3g2) - Dose Response

GABAa5 ver

icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) data and applications:
Cells were kindly provided by Millipore.

Pharmacology on GABAA α5β3γ2 as recorded on the SyncroPatch96. Raw data traces of one exemplary cell using increasing GABA concentrations (A) or increasing Bicuculline concentrations and a subsequent washout (B).

 

 

 

GABAA Receptor (a5b3g2) - Antagonists

GABAa5 Antagonists small

icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) data and applications:
Cells were kindly provided by Millipore.

Mean concentration response curves for Bicuculline, IC50 = 327 nM (n = 14); for a5IA IC50 = 933 pM (n = 11),  maximum block was 45% at 100 nM; for FG7142 IC50= 2.5 mM (n = 9), maximum block was 64.3% at 10 μM; for MRK016 maximum current inhibition was 52% at 1uM, IC50= 1.02 nM (n = 15).

Application Notes

GABAA a5b3g2 - "Characterization of hGABAA α5β3γ2 on Nanion's SyncroPatch 96"

icon sp96   SyncroPatch 96 application note, (a predecessor model of the SyncroPatch 384PE)   logo pdf   (0.5 MB)
Cells were kindly provided by Millipore.

GABAA: Further Readings

GABAA receptors - Investigation of Modulators

p37 1 modulatorsicon pl   Patchliner data and applications:
Cells were kindly provided by AstraZeneca.

The top images show dose dependent block of GABAA currents by bicuculline. The IC50 was determined as 1.2 ± 0.2 μM (n=11). The lower graph shows the positive modulation of glycine activation of hGlyRα1. Here, six co-applications of 20 μM glycine and increasing concentrations of a positive modulator are shown.

GABAA Receptor - Stacked Application Technology

p36 3 timedIntervicon pl   Patchliner data and applications:

Ligand gated ion channels often display receptor desensitization. A method was developed to minimize ligand exposure times and intervals between ligand exposures. The pipette first aspirates buffer, then compound. When expelling this stack, the cell is first exposed to ligand and then buffer. Exposure times as low as 400 ms are possible with this method. A GABA dose response curve, aquired in this manner, is shown on the left.

GABAA Receptor - Modulator Diazepam

Diazepam

 icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) data and applications:

Shown is a raw current response of a HEK293 cell expressing GABAA receptors to initial exposure to GABA, followed by joint exposure to GABA and diazepam (as indicated). Solutions were  stacked (layered) in the pipette to achieve brief exposure times.

GABAA Receptor - Dose Response Curves

0SyncroGabaCRC

icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) data and applications:

Increasing concentrations of GABA were applied to each cell. Cells were from frozen stocks and were prepared for a FliPR experiment running alongside.
The corresponding online analysis of this experiment is shown in the data set "GABAA - CRC Analysis".

 

GABAA Receptor - Currents in iCell Neurons

GABA Bicuculline neurons

icon pl   Patchliner data and applications:
Cells were kindly provided by Cellular Dynamics.

Activation of GABAA receptor currents by 30 μM GABA and partial block of the current response by 1 μM bicuculline. Bicuculline was pre-applied for at least 30 s before co-application with GABA (30 μM). Approximately 50% of the current was blocked by 1 μM bicuculline.

GABAA Receptor - CRC Analysis

0GABACRC OLA

icon sp96   SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) data and applications:

Online analysis results of the dose response curves were obtained with a few mouse clicks. The color code of the online analysis helps to visualize different concentrations applied.

The corresponding data traces is shown in the data set "GABAA - Dose Response Curves".

2016 - Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module

icon sp96  SyncroPatch 384PE publication in Journal of Lab Automation (2016)

2015 - Novel screening techniques for ion channel targeting drugs

icon pl  Patchliner,   icon sp96   SyncroPatch 384PE and   Icon CE   CardioExcyte 96 publication in Channels (2015)

2014 - Crystal structure of a human GABAA receptor

icon pap  Port-a-Patch publication in Nature (2014)

2012 - Characterizing Human Ion Channels in Induced Pluripotent Stem Cell-Derived Neurons

icon pl   Patchliner publication in Journal of Biomolecular Screening (2012)

2009 - Port-a-Patch and Patchliner: High fidelity electrophysiology for secondary screening and safety pharmacology

icon pap  Port-a-Patch and   icon pl   Patchliner publication in Combinatorial Chemistry & High Throughput Screening (2009)

2007 - Automated ion channel screening: patch clamping made easy

icon pap  Port-a-Patch and   icon pl   Patchliner publication in Expert Opinion Therapeutic Targets (2007)

2006 - Microchip technology for automated and parallel patch clamp recording

icon pap  Port-a-Patch and   icon pl   Patchliner publication in Small Journal (2006)

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