Application Note

Channel:Cells:
Tools:hGABAA a5b3g2
HEK293Port-a-Patch

Activation and inhibition of GABAA a5b3g2
receptors on the Port-a-Patch
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells were kindly supplied by Merck Millipore.
SummaryResults

Gamma aminobutyric acid type A (GABAA) receptors

GABAA receptors with the subunit combination a5b3g2

receptors in the mammalian central nervous system(Figure 1). Currents started to activate at concentrations(CNS). They are opened by GABA allowing the pas-above 3 µM and reached maximum at a concentration

sage of chloride ions across the membrane. GABAAof 30 µM. A concentration response curve was construct-including benzodiazepines, barbiturates, neuroactive

culated to be 10.8 ± 0.2 µM (n = 8), in good agreement

are the most important inhibitory neurotransmitterchannels are modulated by a variety of different drugs
steroids, anesthetics, and convulsants1. The receptors areheteropentameric and depending on the subunit combination, they exhibit different electrophysiological andpharmacological properties2. Six a-, three b-, three g-,

were activated by increasing concentrations of GABA

ed for an average of 8 cells (Figure 1B). The EC50 was cal-with the literature for cloned GABAA receptors expressing
the a5-subunit6.
AB

one d-, one e-, one p-, one θ- and three r-subunits have

GABA
1.0 EC = 10.8 ± 0.2 µM
50
0.8 (n = 8)

been cloned, including splice variants of some of theseble with two a, two b, and one g subunit, with alternating a and b subunits connected by a g subunit1. GABAA
receptors play a critical role in regulating excitabilityof the brain, anxiety, vigilance, as well as learning andmemory2. The a5 subunit is highly expressed in the hippocampus and olfactory bulb and expressed in low levels
in other brain regions including the cortex, subiculum,
hypothalamus, sympathetic preganglionic neurons, andamygdala5. GABAA receptors containing the a5 subunit cluster at both extrasynaptic sites as well as synaptic sites thus contributing to tonic currents and synaptic
GABA-ergic neurotransmission5. a5 -containing receptors
exhibit unique physiology and pharmacology and theyare potential pharmacological targets for the treatmentof neurodevelopmental disorders, depression, schizophrenia, and mild cognitive impairment.
The Port-a-Patch with External Perfusion System was used
to record a5b3g2 receptors expressed in HEK293 cells.

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1 µM GABA
3 µM GABA
10 µM GABA
30 µM GABA

I/Imaxsubunits3,4. Functional GABAA receptors typically assem-0.6
0.4
0.2
0.0

0.5 nA11s2

3 4 5 6 78210[GABA (µM)]

3 4 5Figure 1: A Whole cell current responses from HEK293 cells expressing a5b3g2
by increasing concentrations of GABA. Shown are current responses froman example cell with perfusion of external GABA at the concentrations
indicated for 2 s. Following this, GABA was washed away by external
recording solution. Holding potential was -80 mV. B Concentration
response curve for an average of 8 cells was constructed and is shown.
EC50 = 10.8 ± 0.2 µM (n = 8).

Application NoteGABAA currents mediated by a5b3g2 could be repetitively

GABAA receptors with the subunit combination a5b3g2

in peak amplitude was observed between the first andcompetitive antagonist, bicuculline, when co-applied

second application of GABA (14% reduction from -525 ±with GABA (Figure 3).

activated by 1 mM GABA (Figure 2). A small decrease

were activated by 30 µM GABA and blocked by the91 pA to -455 ± 65 pA). Following this, repeated application of GABA resulted in identical peak current amplitudes (< 3% difference compared with the previous amplitude).

0-100

0.2 nAIGABA (pA)

100 pA-200

30 µM GABA
30 µM GABA + 30 µM bicuculline

2s-3000.5 s-400

Figure 3: a5b3g2 GABAA receptors activated by 30 µM GABA were blocked
by 30 µM bicuculline when co-applied.

-500

In conclusion, GABAA receptors with the subunit comn=5-600
1243
Trace number

56Figure 2: Traces from an example cell showing repetitive activation by
1 mM GABA (top) and the corresponding peak amplitudes for an averageof 5 cells (bottom).

bination a5b3g2 were reliably recorded on the Port-aPatch with External Perfusion System. Perfusion of GABA
was triggered automatically by the amplifier and repetitive activation of GABAA receptors was possible using ahigh concentration of GABA. In this way, both inhibitors,
such as bicuculline, and other modulators (not shown)
could be co-applied and characterized using the Porta-Patch.

References

Methods

1. Sieghart, W. 2006. Advances in Pharmacology. 54(5):231-263
2. Sarto-Jackson, I. & Sieghart, W. 2008. Mol. Memb. Biol
25(4): 302-310
3. Simon, J., et al., 2004. JBC. 279(40): 41422–41435
4. Olsen, R.W. & Sieghart, W. 2009. Neuropharm. 56(1):141–148
5. Jacob, T.C. 2019. Front. Mol. Neurosci. 12:179.
doi: 10.3389/fnmol.2019.00179
6. Petroski, R.E., et al. 2006. JPET. 317(1): 369-377

Cells
HEK293 cells stably expressing a5b3g2 GABAA receptors
were kindly provided by Merck Millipore.
Cell culture
HEK293 cells stably expressing a5b3g2 GABAA receptors
were cultured using standard culture conditions for thePort-a-Patch.
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for the Porta-Patch. Currents were elicited by 2 s perfusion of GABA
at different concentrations. A continuous holding
potential of -80 mV was used. Bicuculline was preincubated for 2 mins prior to co-application with GABA.

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