Mechanical and Pharmacological Activation of Piezo1 Channels Characterized by High Throughput
Electrophysiology
Andrea Brüggemann1, Nicoletta Murciano1,2, Maria Giustina Rotordam1, Markus Rapedius1, Nadine Becker1, Melanie J. Ludlow3, Gregory Parsonage3, Kevin Cuthbertson3,
Richard Foster3, David J. Beech3.
1 Nanion Technologies, Munich, Germany, 2 Theoretical Medicine and Biosciences, Saarland University, Homburg, Germany, 3 Univ Leeds, Leeds, United Kingdom.

PIEZO1 currents activated by increasing pipetting speeds
mPiezo1-TREx

CDifferent pipetting volumes

HEK-TRExhPiezo1-TREx

mPiezo1-TREXHEK-TREx✱✱✱✱✱✱✱✱

ns000-500-500-500-1000-1500

I (pA/pF)

B-1000-1500-1000-1500Figure
6. Correlation between currents
elicited by MS and MS-2000+ Yoda1 (A) Before-2000
-2000
Yo
da
1

M

S

Yo
da
1

SM

M

Yo
da
1

S-2500 values elicited by MS and-2500
after-2500
charts comparing current density
MS + Yoda1 at theFigure 3: Averaged current values over time (top) of PIEZO1 activated by transient solution addition at threedifferent pipetting speeds, 40 µl/s, 60 µl/s and 110 µl/s, for a total of six consecutive liquid additions in the absence(MS) (grey columns) and presence of 10 µM Yoda1 (MS + Yoda1) (light blue columns). Dots indicate current values
from n averaged individual cells at the indicated pipetting speed. Averaged raw traces (bottom) activated bytransient solution addition at three different pipetting speeds, 40 µl/s (light blue traces), 60 µl/s (blue traces) and 110µl/s (dark blue traces), in the absence (MS) and presence of 10 µM Yoda1 (MS + Yoda1) from hPiezo1-TREx(n=49/145) (A), mPiezo1-TREx (n=61/143) (B), and HEK-TREx cells (n=0/57) (C). Scale bars 500pA/2s for all the figures.

+SMMMSS++maximum speed of 110 µl/s from hPiezo1-TREx (left) (n=209/758), mPiezo1-TREx(middle) (n=299/691) and(right) (n=27/694), (N=15). Comparisons of medianhPiezo1-TREx

mPIEZO1-TREx

response magnitudes for all charts were made using Wilcoxon matched-pairs test as1000

1000MS (pA/pF)

Under voltage-clamp we were able to show reliable quantification of
PIEZO1 activation by fluid flow, Yoda1 (a small-molecule PIEZO1agonist) and a Yoda1 analogue.

hPiezo1-TREx

I (pA/pF)

The pharmacology of the PIEZO1 channels is in its infancy. Here wesought high throughout methodology for investigating small-molecule
modulation in combination with mechanical stimulation.
A bottleneck in PIEZO drug development has been the lack ofmechanical stimulation in automated patch clamp. Here we show
how the optimization of pipetting parameters and the modificationof the NPC-384 chip of the SyncroPatch 384 lead to the possibility tomechanically stimulate PIEZO1 channels using high throughput
electrophysiology. Data of mouse and human PIEZO1 channels
expressed in HEK T-REX 293 cells activated by either mechanical orchemical stimuli will be shown as well as the combination of bothmethods.

AMS (pA/pF)

PIEZO channels are mechanically-activated cation channels thatplay important roles in biological functions including touch,proprioception, shear stress and stretch sensation as well as bloodpressure regulation. Mutations in Homo sapiens PIEZO1 channels areassociated with anemia, malarial resistance, lymphatic dysplasia andvaricose vein disease, suggesting important red blood cells andvascular roles in humans.

Correlation between currents elicited by MS andMS + Yoda1

I (pA/pF)

Abstract

the data(****p<0,0001) (B) Scatter charts showing
100 did not fit a parametric distribution.
1001010the correlation
between current density values
elicited by MS and MS + Yoda1 from1

To our knowledge, this is the first time that mechanical stimulation ofPIEZO channels in a high throughput planar patch clamp system has
been shown. The possibility of comparing and combining
mechanical and chemical stimulation in a high throughput
electrophysiologicalassay
facilitates
thebiophysicalandpharmacological studies of PIEZO channels.

0.1

1

Current density and percentage of responding cells evaluation at three different
speeds of addition

10100

1000 10000

1r=0,7217; mPiezo1-TREx, r=0,6818)

MS + Yoda1 (pA/pF)

10

100

Transient solution addition on the SyncroPatch 384

maximum speed of 110 µl/s from hPiezo1-TREx (left) (n=209/758), mPiezo1-TREx

1000 10000

MS + Yoda1 (pA/pF)(middle) (n=299/691) and HEK (right) (n=27/694), (N=15). Comparisons of median
response magnitudes for all charts were made using Wilcoxon matched-pairs test as

Figure 5: (A) Before-after charts comparing current density values elicited byMS and MS + Yoda1 at the maximum speed of 110 µl/s from hPiezo1-TREx(left) (n=209/758), mPiezo1-TREx (middle) (n=299/691) and HEK–TREx cells(right) (n=27/694), (N=15). Comparisons of median response magnitudes for
all charts were made using Wilcoxon matched-pairs test as the data did not
fit a parametric distribution. (****p<0,0001) (B) Scatter charts showing thecorrelation between current density values elicited by MS and MS + Yoda1
from hPiezo1-TREx (left) (n=61) and mPiezo1-TREx cells (right) (n=76).
(hPiezo1-TREx, r=0,7217; mPiezo1-TREx, r=0,6818)

This work was supported by research grants from the Wellcome Trust,
British Heart Foundation and has received funding from the EuropeanUnion’s Horizon 2020 research and innovation program.

after charts comparing current density values elicited by MS and MS + Yoda1 at the1hPiezo1-TREx (left) (n=61)
and mPiezo1-TREx cells (right) (n=76).
r=0.7217
r=0.6818 (hPiezo1-TREx,0.1

Figure 6. Correlation between currents elicited by MS and MS + Yoda1 (A) Before-

the data did not fit a parametric distribution. (****p<0,0001) (B) Scatter charts showing
the correlation between current density values elicited by MS and MS + Yoda1 from
hPiezo1-TREx (left) (n=61) and mPiezo1-TREx cells (right) (n=76). (hPiezo1-TREx,r=0,7217; mPiezo1-TREx, r=0,6818)

Figure 3. Position of the cell relative to the pipette. (A) The cell is close tothe pipette. Averaged traces for PIEZO1- mediatedcurrents activated bymPiezo1-TREx

Close to the pipette

Percentage of responding cells at 110 µl/s speedtransient solution addition in the absence (MS) (dark blue traces) and presenceof 10 µM Yoda1 (MS + Yoda1) (blue traces) from mPiezo1-TREx cells (n=147).
(B) The cell is distant from the pipette. Averaged traces for PIEZO1-mediated100MS
MS + Yoda1
activated by transient solution addition in the absence (MS) (darkblue traces)✱✱100pA✱✱✱✱✱✱✱✱✱✱✱✱

ns80% responders

Figure1: Transientaddition
the SP384.
Schematic
illustration of aand presenceof 10solutionµM Yoda1(MS +onYoda1)(blue traces)
frommPiezo1-TREx
n=147
1s chips. The transient addition
cross section of one well from NPC – 384
deliversa small
volumeof solution
to the cell (1 and 2) and uses ancells (n=53).
Scalebars 100pA/1s
forlocallyall the figures.
aspiration step (3, dashed arrows) to recover the dispensed volume.

60 currents elicited by MS and MS + Yoda1 (A) BeforeFigure 6. Correlation between
after charts comparing current density values elicited by MS and MS + Yoda1 at theEffect of the position of the cell relative to the pipettemaximum speed of 110 µl/s from hPiezo1-TREx (left) (n=209/758), mPiezo1-TREx

mPIEZO1-TREx
Distant from the pipette
BmPiezo1-TREx
Close to the pipette
Figure 3. Position of the cell relative to the pipette. (A) The cell is close(middle) (n=299/691) and 20
HEK–cells (right) (n=27/694), (N=15). Comparisons ofto the pipette. Averaged traces for PIEZO1- mediated currents activated bytest as the data did not fit a parametric distribution. (****p<0,0001) (B) Scatter chartsMSMS
transient solution addition in the absence (MS) (dark blue traces)MSandpresence+ Yoda1
MS + Yoda1

showing the correlation between current density values elicited by MS and MS + Yoda1

100pAmPiezo1-TREx
A Close to the pipette40from hPiezo1-TREx (left) (n=61) and mPiezo1-TREx cells (right) (n=76). (hPiezo1-mPiezo1-TREx
Distant from the pipette

Figure 7: (A) Averaged raw traces activated by transient solution addition at
110 µl/s elicited by three different pipetting volumes, 20 µl (dark blue traces),
40 µl (blue traces) and 50 µl (light blue traces) in the absence (MS), (left) andpresence of 10 µM Yoda1 (MS + Yoda1) (middle, right) from cells mPiezo-TREx
cells (left, middle) (20 µl n=257/590; 40 µl n=378/590; 50 µl n=427/590; N=5)
and hPiezo1-TREx cells (right) (20 µl n=222/645; 40 µl n=359/645; 50 µln=420/645; N=5). Scale bars 500pA/1s. (B) Current density values and AUCvalues (C) plotted against three different pipetting volumes, 20 µl (dark bluesquares), 40 µl (blue squares) and 50 µl (light blue squares) elicited by MS only
(B left) and in the presence of 10 µM Yoda1 (MS + Yoda1) (B, middle, right)
from mPiezo-TREx cells (left, middle) and hPiezo1-TREx cells (right). Each data
point (square) represents the current density or AUC of a single cell. Data areshown as median with 95% CI indicated in black. (D) Percentage of mPiezo1TREx cells (N=5) responding to activation by MS +10 µM Yoda1 plottedagainst three different pipetting volumes. Each data point represents anindependent experiment with median with 95% CI indicated in black.
Multiple comparisons of median response magnitudes for all figures weremade using a Kruskal-Wallis ANOVA with Dunn’s post-hoc test as the datadid not fit a parametric distribution. (*p<0.05, **p < 0.01, ****p<0,0001)

median response magnitudes for all charts were made using Wilcoxon matched-pairs

Yoo1oda1-T
TRR
1ExExMmM
SSP+
Hi
E K ezYooda-T1TR
R
1
Ex
Ex
M
M
SS+
H
EK Yo-T da1
REx
M
S+

hPiez

M

S

mediated activated by transient solution addition in the absence (MS) (darkFigure 2: (A) The cell is close to the pipette. Averaged traces for PIEZO1mediated currents activated by transient solution addition in the absence
blue traces) and presence of 10 µM Yoda1 (MS + Yoda1) (blue traces) from(MS) (dark blue traces) and presence of 10 µM Yoda1 (MS + Yoda1) (bluetraces) mPIEZO1-TREx
from mPiezo1-TREx cells (n=147). (B) The cell is distant from themPiezo1-TREx cells (n=53). Scale bars 100pA/1s for all the figures.
pipette. Averaged traces for PIEZO1-mediated activated by transient
Distant
frominthesolution
additionthe pipetteabsence (MS) (dark blue traces) and presence of10 µM Yoda1 (MS + Yoda1) (blue traces) from mPiezo1-TREx cells (n=53).
Scale bars 100pA/1s for all the figures.

ezTREx, r=0,7217; mPiezo1-TREx, r=0,6818)

Pi(B) The cell is distant from the pipette. Averaged traces for PIEZO1-Figure 4: Current density values plotted against three different pipetting speeds, 40 µl/s (light blue squares), 60 µl/s
(blue squares) and 110 µl/s (dark blue squares) elicited by MS only (A, B left) and in the presence of 10 µM Yoda1(MS + Yoda1) (A, B middle) from hPiezo1-TREx (A) and mPiezo1-TREx cells (B). Each data point (square) represents
the current density of a single cell. Data are shown as median with 95% CI indicated in black. Percentage of
hPiezo1-TREx (N=10) (A, right) and mPiezo1-TREx cells (N=11) (B, right) responding to activation by MS +10 µM Yoda1
plotted against three different pipetting speeds. Each data point represents an independent experiment withmedian with 95% CI indicated in black. Multiple comparisons of median response magnitudes for all figures (A, B)were made using a Kruskal-Wallis ANOVA with Dunn’s post-hoc test as the data did not fit a parametric distribution.
(*p<0.05, **p < 0.01, ****p<0,0001)
hPiezo1-TREx cells (40 µl/s n=108/1361; 60 µl/s n=242/1361; 110 µl/s n= 529/1361; N=10);
mPiezo1-TREx cells (40 µl/s n=120/1429; 60 µl/s n=230/1429; 110 µl/s n= 489/1429; N=11).

m1so1-TRExn=147

1sezof 10 µM Yoda1 (MS + Yoda1) (blue traces) from mPiezo1-TRExcells (n=147).
n=53hPi100pA

0Figure 6: Percentage of hPiezo1-TREx (N=15), mPiezo1-TREx (N=15) and HEKTREx cells (N=15) responding to activation by MS +10 µM Yoda1. Each data
point represents an independent experiment with median with 95% CI
indicated in black. Multiple comparisons of median response magnitudes
were made using one-way ANOVA with Tukey’s post-hoc test.
(**p < 0.01, ****p<0,0001)

Figure 6. Correlation between currents elicited by MS and MS + Yoda1 (A) Before-Discussion

after charts comparing current density values elicited by MS and MS + Yoda1 at theIn this study, we set out to develop an electrophysiological highthroughput
mechanically
cells expressing
themaximum
speed approachof 110 µl/s tofrom
hPiezo1-TRExstimulate(left) (n=209/758),
mPiezo1-TREx
mechanosensitive ion channel PIEZO1 by using the SyncroPatch 384.
(middle)(n=299/691)
HEK–cells (right) (n=27/694),(N=15). Comparisonsof median
To optimize
theandmechanosensitive
current response,
we considered
three parameters: 1) the position of the cells relative to the pipette
response magnitudes for all charts were made using Wilcoxon matched-pairs test as thetips, 2) the pipetting speed, and 3) the applied volume of solution.
Thethatdistribution.
it is possible
to activate
Piezo1currents
data
did results
not fit ashow
parametric(****p<0,0001)(B) Scattercharts
showingvia
themechanical stimulation and combine it with chemical stimulation ascorrelation
well. between current density values elicited by MS and MS + Yoda1 from
We believe that this is a great starting point for a direct HTS assay for
hPiezo1-TREx (left) (n=61) and mPiezo1-TREx cells (right) (n=76). (hPiezo1-TREx,
studying the Biophysical and chemical properties of mechanosensitive
channels. r=0,6818)
r=0,7217;
mPiezo1-TREx,