May‘08

Application Note
Channel:GABAA (α1β2γ2)

Cells:

HEK293Tools:Patchliner©

Investigating the pharmacology of GABAA (α1β2γ2)receptors expressed in HEK293 cells.
The electrophysiology team at Nanion Technologies GmbH, Munich.

SummaryResults

Using the Patchliner platform, the pharmacology of
γ-amino butyric acid (GABA), β-alanine and bicuculline were investigated on GABAA-receptors expressed in HEK293 cells.
The GABAA receptor family is the most importantclass of inhibitory ion channels involved in synaptictransmission. GABAA receptors are selectively permeable to monovalent anions. They constitute animportant therapeutic target for drugs affecting
anxiety, sleep and muscle relaxation.
In this study, the cells were exposed to compoundfor 30 s, followed by a 60 s wash step. Solution exchange around the cell was fast, in the order of 50 ms
for saturating concentrations, see Fig. 1. Complete
and rapid switching is important when investigating
ligand gated ion channels, since the response isoften very fast and most receptors desensitize. For
rapidly desensitizing ion channels, fast compoundapplication is crucial, so that the entire ion channel
population is exposed to maximum concentration
before entering the desensitized state. Desensitization and recovery kinetics vary from milliseconds totens of minutes, depending on receptor type andsubunit composition as well as exposure time and/or compound concentration.
Recordings were made in the whole-cell configuration, using the Patchliner (4 amplifier channels).
Expected pharmacology was obtained for the investigated compounds.

GABA was rapidly added to the cells with a total
exposure time of 30 s. Wash intervals with control
solution were 60 s. Figure 1 shows the concentration
dependent activation of GABAA-receptors by application of increasing concentrations of GABA (0.3,1, 3, 10, 30, 100 µM). Peak current amplitudes werenormalized against the first saturating response (Figure 2). The EC50 was determined as 2.7 ± 0.5 µM
from the Hill plot. The error bars show the standard
error of the mean.

nan]i[onFigure 1: Whole-cell currents evoked by GABA-applications (increasing concentrations) for the determination of the EC­50. Application is fast, well below
20 ms for the higher concentrations (insert).

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Compound application is fast. In these experiments
it ranged from 50 ms to 100 ms (mean 78 ms) for
the application of 10 µM of GABA, and was fasterfor concentrations higher than 10 µM, see insert Figure 1.
The pharmacology of the agonist β-Alanine wasalso investigated. The exposure time to β-Alanine
was 30 s followed by a 60 s wash step. The peakcurrent amplitudes were normalized against thefirst saturating response and plotted against logconcentration (Figure 3). The EC50 value was determined as 670 ± 70 µM. As expected, the potency of
β-Alanine is at least two orders of magnitude lower
than that of GABA.

Figure 3: (A) The EC50 for β-Alanine was determinedby the application of increasing concentrations of
β-Alanine with intermittent wash steps. (B)The IC50
for bicuculline was determined by the appliationof control (10 µM GABA) followed by co-application of 10 µM GABA with increasing concentrationsof bicuculline.

The potency of the anatgonist bicuclline was investigated. Increasing concentrations (0.3, 1, 3, 10 µM)
of bicuculline were co-applied with 10 µM GABA,for determination of the IC50 value. The values forthe peak amplitudes were normalized against thecontrol (10 µM GABA - Figure 3). The IC50 value wasdetermined from the Hill plot as 1.14 µM, which corresponds well to the literature (1 -3 µM).

Figure 2: The EC50 value for GABA was determinedby plotting the normalized peak current amplitudes against concentration (log scale). The obtained value corresponds well with the literature.

References

Methods

1.
Hille, B., Ion channels of Exitable Membranes, 2001, Third Edition, Sinauer Press.
2.
Lu, J., Greco MA., 2006, Sleep circuitry andthe hypnotic mechanism of GABAA drugs, J. Clin.
Sleep Med. 15:19-26
3.
Cosette, P., Lidong, L., Brisebois, K., et al.
2002, Mutation of GABA1 in autosomal dominant
form of juvenile myoclonic epilepsy, Nature genetics, 31:184-189
4.
Cotrell, G.A., LambertJ.J., Peters, J.A., 1987,
Modulation of GABAA receptor activity by alphaxalone, Br. J. Pharmacol., 90(3):491-500

Cells
HEK293 cells stably expressing GABAA were used.
Cell culture
Cells were cultured and harvested according toNanion‘s standard cell culture protocol.
Electrophysiology
Whole cell patch clamp recordings were conducted according to Nanion’s standard procedure
for the Patchliner©. Cells were clamped to a holding
potential of -80 mV. Compounds solutions were diluted in external recording solution, prepared daily
from frozen stocks.

nan]i[onNanion Technologies GmbH
Erzgiessereistr. 480335 Munich, Germany

phone +49 - 89 - 2189 979 72
fax +49 - 89 - 2189 979 60
http://www.nanion.de - info@nanion.de