Application NoteTopic:
GPCRs (Y4R, H1R)

Profiling the pharmacology ofGPCRs by time-resolved impedance
measurements

Cells:
U373 endogen. H1R,CHO overexpr. Y4RTools:
AtlaZ

Julia Erl and Joachim Wegener, University of Regensburg,
Stefanie Michaelis, Fraunhofer EMFT, Regensburg.

Summary
G-protein-coupled receptors (GPCRs) are extensively studied
drug targets in medicinal chemistry and pharmacology. The
human genome encodes almost 900 GPCRs, which accountfor approximately 4 % of the genome, hence it’s not surprisingthat they play roles in numerous physiological processes. It isestimated that about 40 % of all drugs act through GPCRs.
Dysfunctions in these receptors have been linked to various
severe diseases, including diabetes, allergies, depression, andcertain forms of cancer, among others 1,2,3,4.
Traditional screening methods to study GPCR pharmacology
involve genetically engineered cells with overexpressed
receptors, focusing on ligand-GPCR binding and theirdownstream signaling. Non-invasive, label-free impedance
measurements offer real-time monitoring of GPCR activation

does not directly modulate second messengers, like cAMP orIP3 (Figure 1); instead, it leads to the activation of Rho family

GTPases, such as RhoA, which regulate cytoskeletal dynamics,
cell migration, and gene expression.
This Application Note demonstrates the in vitro characterizationof GPCR pharmacology, covering agonist and antagonist
mode of action, dose-response relationships, and theinvolvement of signal transduction cascades. Experimentally,cells expressing H1R or Y4R are cultured on planar gold-film
electrodes integrated into standard cell culture dishes. Here, theplanar gold-film electrodes embedded in a 96-well plate format
are used as growth substrate for the adherent cells and as asensor to monitor the cell response. H1R is a GPCR that is known
to activate phospholipase C (PLC) via Gq, whereas Y4R belongs(agonist/antagonist mode) in target cells with endogenous
receptor expression or engineered cells lines overexpressing thereceptor. Heterotrimeric G-proteins transmit receptor activation
into intracellular signaling cascades. Based on the amino acidsequences of their individual Gα-subunits (Gs, Gq, Gi/o, G12/13),a distinct signaling pathway is triggered. Here, we demonstrate

GPCR

Ligandactivated GPCRs

Autoantibodyactivated GPCRs

the usefulness of impedance-based assays to unravel GPCR
pharmacology by example of two of the three signaling
pathways involving second messengers: (i) the Histamine H1
Receptor (H1R), which couples through the Gq-protein, and (ii)the Neuropeptide-Y Receptor 4 (Y4R), which couples throughthe Gi-protein. Unlike the other pathways, the G12/13 pathwayGiGqGs

G12/13cAMPIP3, Ca2+cAMPRhoGEFs

Signaling Signaling Signaling Signaling

Morphological changes
Figure 1: Why is label-free impedance sensing so
attractive? Different GPCR-dependent signaling
pathways induce morphological changes of cells.
Independent of which pathway being active, thesechanges can be measured via impedance.

Impedance provides a holistic view on cell signalingto the neuropeptide Y receptor family coupled to Gi.

was recorded at 10 frequencies between 100 Hz and 100 kHz

For the Y4R, we used the endogenous agonists hPP (humanequally spaced on a logarithmic scale. The stock solution ofpancreatic peptide) and the Synthetic Agonist 1 (SA1). CHO

the agonist Histamine (Histamine Dihydrochloride) was 20 mM

cells overexpressing the Y4R were exposed to these agonists.

in L-15 medium, stored at – 20 °C and diluted in L-15 medium

Impedance measurements as performed here reveal changes

according to the required working concentration. As a control

in cell morphology.

experiment, the antagonist Mepyramine (Mepyramine Maleate,
M = 401.5 g/mol; stock was 10 mM in L-15 medium, stored at

The strength of the AtlaZ assay approach with regards to– 20 °C) was added to wells 20 min prior to Histamine stimulation.

GPCR as a target is its (i) time resolution, (ii) throughput, (iii)

Final concentrations of Histamine were 0 µM, 0.03 µM, 0.1 µM,

the fact that it is label-free and (iv) independent of genetic

0.3 µM, 0.6 µM, 1 µM, 3 µM, 10 µM, 30 µM. In antagonist mode,engineering. The technology is applicable to primary cultures

the cells were pre-incubated with 0.03 µM Mepyramine foror finite cell lines with endogenous receptor density 5. A crucial

20 min.

advantage over standard assays that rely on labels for optical

Data presented in Figure 2 B reveal a concentration-dependentor radiometric detection, is the continuity of cell monitoring.

response of the cells upon Histamine addition. The impedance

Endpoint assays using dyes or labels just report on the status ofincrease mirrors the cell shape change that is associatedthe cells at a single time point but neglect the kinetics of the cellwith the intracellular, Gq-mediated signaling cascade. Theresponse.

impedance increases steadily after stimulation, runs througha maximum about 10 min after compound addition before

Results

it slowly returns to baseline conditions. This profile is similar toIn this study, we used U373 cells endogenously expressing thethat the stimulation is predominantly mediated by the Ca2+/IP3

profiles observed earlier 6 and is a strong experimental indicator

Histamine H1 Receptor (H1R) or CHO cells overexpressing theNeuropeptide-Y Receptor 4 (Y4R).

dependent signaling cascade. The EC50 value was estimated tobe 689.5 nM (Figure 2 D). Mepyramine blocked or reduced theHistamine effect dependent on Histamine concentration (dataU373 cells endogenously expressing H1R

not shown).

U373 is a human glioblastoma astrocytoma cell line derived
from a malignant tumor by explant technique. U373 cells were

CHO cells overexpressing Y4R

seeded in culture medium at a density of 100.000 cells/cm2.

The NPY receptor 4 is coupled to the Gi/o-protein that reduces

Cell adhesion was monitored over the next 20 h (Figure 2A).

the activity of the cAMP-producing enzyme adenylate

Medium was exchanged 2 days after cell seeding. On day 3,cyclase. With cAMP concentrations being low in resting cells,

U373 cells were stimulated with Histamine dissolved in L-15

the activation of the Gi/o pathway is experimentally hard tomedium (CO2-independent). At the beginning of the assay,

catch unless adenylate cyclase is experimentally stimulated

cells were allowed to adapt to fresh L-15 medium (100 µL/well)before. A well-established, receptor-independent inducer ofwithin 2 h prior to stimulation. The recording mode used here

adenylate cyclase is forskolin. To find an appropriate forskolin

was “multiple frequencies” i.e. the impedance is recorded

concentration, CHO Y4R cells were exposed to 0.1 nM, 1 nM,

at several designated AC frequencies. Here, the impedance

10 nM, 100 nM, 1 µM and 10 µM forskolin (Figure 3 A, B). To

BHistamine stimulation

1000
Normalized Cell Signal

Normalized Cell Signal

A

900
800
700
600
0510
Time / h

15

1.2
1.1

1.0
48.5

20

49.5

50.0

Time / hD
Normalized Cell Signal

C49.030 µM
10 µM
3 µM
1 µM
0.6 µM
0.3 µM0.1 µM
0.03 µM
0 µM

Normalized Cell Signal(t = 48:53 hh:min)

1.2

1.3

Figure 2: Time-resolved impedance profiles of

1.2

1.0

1.1

human glioblastoma cells (U373), (A) raw traces

1.0

0.

0µM
03
µ0. M
1µ0. M
3µ0. M
6µM
1µM
3µM
10µM
30µM

0.9

EC50 = 689.5 nM
10-5

10-4
10-3
10-2
log(concentration)

10-1

for adhesion and growth (seeding at time zero,n = 14 wells), (B) normalized traces for stimulationof U373 cells with Histamine at 0.03 μM - 30 μM(normalized to cell signal in L-15 medium before
Histamine addition), (C) bar graph for normalized
cell signal at t = 48:53 hh:mm. (D) EC50 estimated tobe 690 nM. Data are shown +/- SD, n = 6 each.

0.5

0101MnM
10n
10 M
0nM
1µM
10µM
µM

1010nM

8µM

6Time / hEC50 = 635,7 p M

0.0

100.5

02010 µM
1 µM
100 nM
10 nM
1 nM
0.1 nM
0 nM

EC50 stimulation(SA1)
1.0

1040

1.5nM

60nM

increasing SA1 concentrations4MF

constant forskolin concentration

11008EC50 = 734.8 p M

0.010

Normalized Cell Signal

6Time / h0.5

10EC50 stimulation(hPP)

1.0nM

2010 µM
1 µM
100 nM
10 nM
1 nM
0.1 nM
0 nM

1.5nM

404n

D

increasing hPPconcentrations
60E

11n

30

0.

20Time/minnM

10

constant forskolin concentration

10Cn
Mµ10 MµM

EC50 = 220 nM

0.

shown +/- SD, n = 6 each.

0.1 nM

0 nMnM

relationship yielding an EC50 of 636 pM. Data are

0.61.01 nM

Normalized Cell Signal

increasing conc. of SA1, (F) dose-response

1 µM
100 nM

1an EC50 of 735 pM. (E) Stimulation of Y4R with

10 µM

0.8

0hPP, (D) dose-response relationship yielding

1.0

1.5

0.

Stimulation of Y4R with the endogenous ligand

EC50 stimulation (forskolin)

1.2

Normalized Cell Signal

relationship yielding an EC50 of 220 nM. (C)B

forskolin stimulation

1.4

Normalized Cell Signal

increasing forskolin conc., (B) dose-response

A

Normalized Cell Signal

Y4R stimulation in CHO cells, (A) stimulation via

Normalized Cell Signal

Figure 3: Time-resolved impedance profiles ofidentify the kinetics of forskolin effect, a high repetition interval

It is also important to emphasize that impedance-basedof 1 s between sequential impedance readings was chosenmonitoring of GPCR stimulation is sensitive enough to work with(Figure 3 A). We estimated an EC50 of 220 nM (Figure 3 B),

endogenously expressed receptors, like in primary cultures orand the optimal forskolin concentration used in the following

even stem cells. The assays will provide the highest physiologicalexperiments was 400 nM, inducing a maximum effect. This

relevance when no component of the signaling cascade

concentration was applied to pre-stimulate CHO Y4R cells prior

is overexpressed. Moreover, it avoids the need for genetic

to the addition of the endogenous ligand hPP at 0.1 nM, 1 nM,engineering prior to receptor characterization and thereby

10 nM, 100 nM, 1 µM and 10 µM (Figure 3 C, D), or SA1 at 0.1 nM,

reduces the biological safety label when conducting the assay.

1 nM, 10 nM, 100 nM, 1 µM (Figure 3 E, F). The result of the doseresponse analysis is given in Figures 3 D and 3 F, respectively.

Impedance-based cell monitoring is often called a holistic

The CHO cell seeding density in these experiments wasapproach as the impedance integrates over the entire

100.000 cells/cm2. The compounds were added once thecell body without any molecular specificity as, for instance,cell layers were fully established. The impedance signal ofprovided by most label-based assays. As such, it provides anthe cell monolayer was recorded every 120 seconds for both,

unbiased perspective on the cell under study. The lack of

hPP and SA1, and is plotted using the unitless parameter Cell

molecular specificity may require some extra careful assay

Signal (Figure 3). The time course of impedance revealed

procedures and controls to avoid false-positive results. But

distinctly different profiles compared to the H1R in U373 cells,

it unfolds its full strength when orphan receptors are being

indicating that different signaling pathways may provide distinct

studied. The search for a yet unknown endogenous agonist

impedance profiles. However, our data suggests this is not

typically goes along with no information on the couplingalways true as it also depends on the origin of the tissues andscheme of the receptor. Thus, an unbiased assay readoutcell density.

that is independent of the downstream signaling cascadebut sensitive to all of them is the most efficient readout in de-In conclusion, the AtlaZ system offers a highly automatedorphanizing campaigns.

approach capable of studying the pharmacology of various
GPCR signaling pathways using a single platform. In addition,
the AtlaZ system does not require labeled reagents andprovides the cell response to receptor stimulation in realtime. The latter has proven its usefulness, for instance, whenphotoswitchable GPCR agonists 7 or receptor desensitization 8
have been studied. Thus, straightforward assay design, minimalartifacts, and short assay time, contribute to higher and more
efficient throughput provided by the AtlaZ.

Methods
AtlaZ platform
The AtlaZ platform (Nanion) provides quantitative live-cell
analysis by measuring the impedance (Ohm, Ω), displayed
as Cell Signal, of adherent cells grown on 96-well plateswith embedded planar gold-film electrodes (Nanion). The
methodology, Electrical Impedance Spectroscopy (EIS),
provides a unique richness of information from cells. Depending

cells adhere and proliferate (<= 48 h) (1). Upon
Adherent GPCR expressing cellGoldelectrodereaching a plateau phase, spectra for cell-free
Cell Signal

1Figure 4: Workflow of a GPCR assay. Target

and cell-covered electrodes reveal the mostproliferation

sensitive frequency (2) by plotting the ratio of
the impedance magnitude |Z| of electrodes

Proliferation & developmentof a confluent monolayer

cellcovered
cell free

0.1
110100Frequency (kHz)with cells and the impedance |Z| of electrodes
most sensitivefrequency

without. Next, the treatment can be executed(3). The Cell Signal is monitored continuously overtime, revealing the kinetics of stimulus-induced

0.1
1
10100
Frequency (Hz)effects.

concentration

agonist /antagonist
Cell Signal

3IZI cell cvrd / IZI cell free

2IZI (Ohm)Time (h)Time (h)on the data acquisition frequency, the system detects
predominantly the resistive part of cell-cell and cell-matrix
contacts at lower frequencies or predominantly capacitive

Key findings• Our results demonstrate the capability of AtlaZ tocurrents across the cell membranes at higher frequencies ascharacterize the pharmacology of GPCRs and monitor

an indicator for electrode coverage. The latter is the basis for

major G-protein-dependent pathways.

cell adhesion or migration assays. Thus, real-time impedance
data provide insights into various cell phenotypes, such as• AtlaZ allows for cellular research on cell adhesion,proliferation, cytotoxicity and GPCR-mediated signal

cell morphology changes as a result of signaling, proliferation,transduction effects, label-free and in real-time.

lateral migration or cytotoxicity even over prolonged periods oftime.

• Recordings can be performed for up to 6 x 96 = 576 samples
either simultaneously or independently.

GPCR assay
Here, we used the CHO cell line which has been transfected
to overexpress a GPCR (CHO-Y4, Y4R) or a finite cell line withendogenous GPCR expression (U373, H1R). The readout,displayed as the Cell Signal, represents raw impedanceReferences
1.

Lisa A. Catapano et al., BBA, Vol. 1768, 2007, 976-993, https://doi.org/10.1016/j.
bbamem.2006.09.025.

2.

Reimann, F., et al., Diabetologia 59, 2016. https://doi.org/10.1007/s00125-015-3825-z

3.

Schöneberg T., et al, Pharm. & Therap., Vol. 104, 2004, 173-206, https://doi.
org/10.1016/j.pharmthera.2004.08.008.

specific and has to be determined empirically, but for most

4.

Sriram K & Insel P.A. Mol. Pharm. 2018, 93, DOI: https://doi.org/10.1124/mol.117.111062.

electrode types it ranges between 1 kHz and 50 kHz 9. As a5.

Skiba M. et al., Ch. 10 Methods in Cell Biology, Academic Press, 2022, https://doi.
org/10.1016/bs.mcb.2021.12.005.

6.

Lieb S. et al. Pharmac. Research, Vol 114, 2016, https://doi.org/10.1016/j.
phrs.2016.10.010.

the impedance magnitude |Z| of a cell-covered electrode by7.

Wirth U et al., 2023, Angewandte Chemie, https://doi.org/10.1002/anie.202215547

the impedance of a cell-free electrode along the frequency

8.

Stolwijk JA, et al. 2019, Pages 99–108, https://doi.org/10.1093/intbio/zyz010

spectrum (Figure 4). When these ratios are plotted for each9.

Stolwijk JA, et al. Pflugers Arch. 2015 doi: 10.1007/s00424-014-1674-0. Epub 2014 Dec 24.
PMID: 25537398.

values. For these specific recordings we have chosen a 12
kHz recording frequency, which is best suited for these typeof pathway investigations. The optimal frequency is cell-type

general guideline, the sensitive frequency for detection ofchanges in cell layer properties can be determined by dividing

individual frequency, one obtains a bell-shaped curve fromwhich the maximum ratio reflects the frequency with thebroadest range of relative change in impedance (Figure 4), i.e.
the most sensitive frequency.

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