Application Note

Channel:Cells:
Tools:

hGlyRa1
L-tk
Patchliner

Modulators for glycine receptors investigated using the PatchlinerTheelectrophysiology

teamatNanion

Technologies

GmbH,Munich.

Data reproduced by courtesy of AstraZeneca. Cells and drugs were kindly provided by AstraZeneca, Sweden.
Summary
The Patchliner was used to study the pharmacology ofcurves and these are compared in Fig. 1. Concentrations

the hGlyRa1 receptor expressed in a mouse ﬁbroblast cellranging from 10 – 3000 µM glycine were used.

line (L-tk). The Patchliner allows for fast application ofintervals

andwashtimes.

Theseareimportantexperimental parameters when investigating ligand
gated ion channels and their effectors since many ligand
gated ion channels rapidly desensitize. Kinetics and levelof desensitization are determined by ligand concentration,

10 µM glycine
30 µM glycine
100 µM glycine
300 µM glycine
1 mM glycine
3 mM glycine

exposure time, or both. Because of the Patchliner’s rapid
solution exchange combined with brief drug exposure
capabilities

andtimed

applicationintervals,

1 nA

normalized whole cell response

drugs (<50 ms) with precisely controlled application
10080604020010 s0.001

the246 8246 82246 8246 820.01
0.1
Concentration (M)can be minimized and corrected for.
Here we show the use of Nanion`s Patchliner for long (22s) and short (1 s) application of glycine to patch clamped
whole-cells. For the short applications of compound, astacked

solution

application

protocolwasused.

10 µM glycine
30 µM glycine
100 µM glycine
300 µM glycine
1 mM glycine
3 mM glycine

Speciﬁcally, two zones of solutions were aspirated into thepipette used for administration. When applied to the cell,
this results in exposure first to the agonist zone followed bya rapid wash out after 1 s of drug application. In all theprotocols used, pre-incubation of modulators is possible,
as well as washout in the presence of a modulator.

Results
The pharmacology of glycine was investigated on GlyRa1
using two different application strategies. Long and shortapplication times were used to obtain dose response

Download more Application Notes from www.nanion.de1 nA
1snormalized whole cell response

deleterious effect caused by receptor desensitization
1008060402000.001

0.010.1
Concentration (M)Figure 1:
Dose response of glycine. Top: Six concentrations of glycine (10, 30,100, 300, 1000, 3000 µM) were applied to the cells with intermittent wash
steps. Four cells were simultaneously recorded, Vhold = -80 mV. Cells
were exposed to glycine for 22 s followed by a 60 s wash step. Bottom:
A stacked application protocol was also used for 1 s application ofglycine to the cells. The same concentrations were used, Vhold = -80 mV.

Application Note

20 µM Glycine+ 0.03 µM AZ10477650+ 0.1 µM AZ10477650+ 0.3 µM AZ10477650+ 1 µM AZ10477650+ 3 µM AZ10477650+ 10 µM AZ10477650
1 mM Glycine

Saturating whole-cell current responses were obtained
between 300 - 1000 µM and the EC50 was determined toStrychnine
Strychnine +pre-incubation Glycine appplication

be 127 µM. Application of 3000 µM glycine showed a sub-2 nA
1s

Wash in presenceof Strychnine

stantial desensitization and the peak current response did
not reach the amplitudes achieved with 300 and 1000 µM
glycine. When the stacked application protocol was used,

0.2 nA1s

the cells were exposed to glycine for 1 s followed by a 60

100 µM glycine (buffer wash out)
100 µM glycine + 1 µM strychnines wash step. Whole-cell currents elicited by 10 – 3000 µM
glycine are shown in the lower panel of Figure 2. As before, currents start to saturate at glycine concentrations
above 300 µM. However, here the peak current amplitudes evoked by 3000 µM of glycine clearly reach saturating
concentrations, which is not the case for the long applica-

Figure 2:
The effect of positive and negative modulators acting on the GlyRαs wasinvestigated. Speciﬁcally, a positive modulator was co-administered inincreasing concentrations with 20 µM of glycine to investigate the EC50
and degree of potentiation. In addition, the antagonist strychnine wasinvestigated. A pre-incubation step was used.

tions. The EC50, determined from the Hill plot, was 157 µM.

As a final control, 1000 µM of glycine was applied. The

time dependent. It is also clear that desensitization kine-tained.

This indicates that receptor desensitization is exposure-tics are concentration dependent, which is evident fromboth long and short agonist exposures.

expected EC50 value and levels of potentiation were obThe lower panel shows block of the hGlyRa1- mediated current by the competitive antagonist strychnine.

Positive and negative modulators have been investigated

Two control applications of glycine were made, before

using the Patchliner®. In the top panel of Figure 2 whole-and after the strychnine application. Currents were notcell traces are shown, showing potentiation of glycine

regained after the block, so the second control is not

responses. Here, first two control applications of glycine

shown. Strychnine (1 µM) was pre-incubated for 20 s, fol-(20 µM) alone were made followed by co-administration

lowed by the co-administration with 100 µM glycine. Sta-with increasing concentrations of the positive modulator.

cked applications were used to apply the compounds.

References

Cell culture

1. Lynch JW, Callister RJ. 2006. Glycine receptors: a new
therapeutic target in pain pathways. Curr Opin Investig
Drugs. 7:48-53.

Cells were cultured and harvested according to Nanion'sstandard cell culture protocol.
Electrophysiology

2. Barry PH, Lynch JW. 2005. Ligand-gated channels. Trans

Whole cell patch clamp recordings were conducted

Nanobioscience. 4:70-80.

according to Nanion’s standard procedure for thePatchliner. Cells were held at a holding potential of -80

Methods

mV. For the short exposure times, solutions were stacked
in the robotic pipettor. First, wash solution was aspirated

Cells

followed by aspiration of the agonist-containing solution.

L-tk cells stably expressing hGlyRα1 were used.

Wash solutions contained the antagonist in the strychnine experiments. The cells were pre-incubated in strychnine before co-application with glycine.

Nanion Technologies GmbHphone + 49 89 219 095-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960