January `08

Application Note
Channel:Cells:
Tools:GABAA(α1, β2, γ2)
HEK293Patchliner©

Rapid agonist applications in precise intervals
using the Patchliner©
The electrophysiology team at Nanion Technologies GmbH, Munich.

Summary
HEK-cells expressing GABAA-receptors were investigated with the Patchliner using a stacked application
approach for rapid administration of compounds tothe patch-clamped cells.
The GABA receptor family is the most important classof inhibitory ion channels involved in synaptic transmission, and are selectively permeable to monovalent anions. They constitute an important therapeuticarea for drugs affecting anxiety, sleep and muscle
relaxation.
As with most ligand gated ion channels, GABAA exhibit receptor desensitization, which is a common
phenomenon for ligand gated ion channels. Desensitization can be either exposure time dependent orconcentration dependent, or both. Desensitization
and recovery kinetics varies from milliseconds to tensof minutes, all depending on receptor type and subunit composition. For rapidly desensitizing ion channels, it is important that compound application is fast,
so that the entire ion channel population is exposed
to maximum concentration before entering the desensitized state.
Exposure time and application intervals are important factors affecting desensitization and recovery
from desensitization, to minimize deletrious effects orreceptor desensitization.
In the experiments presented here, compounds wereadded to the cells with accurate timing. In addition,a method for brief compound application was developed, allowing 1 s pulses of compound to the patchnan]i[onclamped cells. In this way, receptor desensitization
can be minimized, and because of the timed exposures, effects of desensitization can be accounted
and corrected for.

Results
A method for stacked application was developed.
Here, buffer solution was aspirated, directly followed
by a plug of compound. In this way, the cell was exposed to GABA for 1 s, at an interval of 90 s. Figure 1
shows the replay window of HEKA’s Patchmaster software, showing the application of 1, 3 and 10 µM ofGABA to four patch clamped cells.

Figure 1: Replay window from HEKA, showing the application of GABA (1, 3 and 10 µM) to four cells.

Download more APPLICATION NOTES on www.nanion.deApplication NoteThe current responses obtained from the stacked
applications were compared to normal compoundapplication, using the same concentrations of GABA.
Here, application time was set to 20 s, with an application interval of 60 s.
The long applications were followed by stacked applications using the same concentrations, and as
seen from the peak current responses, application
time and wash time are acceptable for avoiding receptor desensitization.

Figure 2: Comparison of long applications of GABA
and the stacked application protocol. GABA (1, 3, 10µM) was applied in timed intervals.
The effect of the antagonist bicuculline was investigated.
In these experiments, shown in Figure 3, four cells werestimulated 3 times with control applications of GABA,
3 µM. Control applications were followed by three
co-applications of bicuculline and GABA (10 µM, 3µM respectively). Peak current amplitudes were plotted against time, showing fairly consistent whole-cell
responses over an extended time period, and thatthe block is reproducible.

Figure 3: Investigation of bicuculline block. Bicuculline
was co-applied using the stacked solution protocol(2), and compared to control applications of GABA
(1 and 3). Peak current amplitudes were plotted overtime as seen in the lower panel.

References

Methods

1. Hille, B., Ion channels of Excitable Membranes,
2001, Third Edition, Sinauer Press.
2. Lu, J., Greco MA. 2006, Sleep circuitry and the hypnotic mechanism of GABAA drugs, J. Clin. Sleep Med.
15:19-26

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 conductedaccording to Nanion’s standard procedure for thePatchliner©. Cells were clamped to a holding potential of -80 mV. Compounds solutions were diluted inexternal recording solution, prepared daily from frozen stocks.

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

phone +49-89-2189 979 72
fax +49-89-2189 979 60
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