Mechanical stimulation of PIEZO1 channels
using high throughput automated patch clamp
Nicoletta Murciano1,2, Maria Giustina Rotordam1, Markus Rapedius1, Nadine Becker1, Melanie J. Ludlow3, Gregory Parsonage3, Alison
Obergrussberger1, Andrea Brüggemann1, David J. Beech3
1Nanion Technologies, Munich, Germany; 2Theoretical Medicine and Biosciences, Saarland University, Homburg, Germany; 3University of Leeds, Leeds, UK

Contact: ali@nanion.de1

Introduction

PIEZO1 channels are mechanosensitive ion channels that play a pivotalrole in sensing mechanical forces in various cell types. Their dysfunction
has been associated with numerous pathophysiological states including
generalized lymphatic dysplasia, varicose vein disease, dehydrated
hereditary stomatocytosis, and malarial resistance. Given its highphysiological relevance, investigating PIEZO1 is crucial for thepharmaceutical industry that requires scalable techniques to allow for
drug discovery. In this regard, several studies have shown the use of highthroughput automated patch clamp (APC) to explore the function andproperties of PIEZO1 channels in heterologous expression system as well as
primary cells (Rotordam et al., 2018, Parsonage et al., 2022; KaramaticCrew et al., 2023) mainly based on usage of Yoda1, a specific gating
modifier of PIEZO1 channels (Syeda et al., 2015). However, to ourknowledge, a combination of solely mechanical stimulation and highthroughput APC has not yet been available for the study of PIEZO1channels. Here we show PIEZO1-mediated currents activated bymechanical stimulation (M-Stim) on the SyncroPatch 384.

3

Effect of pipetting flow on PIEZO1
responses

Figure 2: Representative PIEZO1 raw
traces of single cells elicited by M-Stimat 40µl/s (light blue trace), 60µl/s (bluetrace) and 110µl/s (dark blue trace)and blocked by GdCl3 (black trace) formPIEZO1(A),hPIEZO1(B)anduntransfected cells (C). Absolute peakcurrent amplitudes of mPIEZO1 (D) andhPIEZO1 cells (G) plotted against
pipetting flow (mPIEZO1: n=123/1440;
N=9; **P=0.0073, ****P<0.0001); hPIEZO1:
n=123/1471;
N=9;**P=0.022,****P<0.0001). Time to peak and T50
values of mPIEZO1 (E-F) and hPIEZO1cells (H-I) plotted against pipetting flow(mPIEZO1: 40µl/s n=4/1467; 60µl/s
n=24/1488; 110µl/s n=88/1440; N=9;hPIEZO1:40µl/s
n=4/1494;
60µl/sn=18/1483; 110µl/s n=103/1471; N=9; ns
P>0.05, *P=0.0201,***P=0.0006). All data
are shown as values of individual cellswith median, and 95% CI indicated inblack, tested for statistical significanceusing a Friedman test (D/G) and KruskalWallis test (E-F/H-I) with Dunn’s post-hoctest.

5Effect of M-Stim combined with Yoda1

2

Mechanical stimulation (M-Stim) on
the SyncroPatch 384

Figure 1: (Left) Schematic illustration of a cross section of one well of an NPC-384 chip. M-Stim delivers
a small volume of solution locally to the cell (1 and 2) and uses an aspiration step (3, dashed arrows) torecover the dispensed volume. (Right) Schematic illustration of the top view of one well of an NPC-384
chip with 1 patch hole (1x) and 4 patch holes (4x). Illustrations were created with BioRender.com.
Abbreviations:n = number of cells for a given experimental condition out of total amount of valid cells
N = number of independent NPC-384 chips
T50 = decay time from peak to 50% of the remaining signal

4Increased current readout using multihole chips

Figure 3: Representative mPIEZO1 (A) and hPIEZO1 (F) raw traces recorded from a 4-hole chip
elicited by M-Stim at 110µl/s. Absolute current values of mPIEZO1 (B) and hPIEZO1 cells (G) recorded
from single-hole (1x, dark blue) and 4-hole (4x, purple) chips (mPIEZO1: 1x, n=148/1910; N=18; 4x,n=579/941; N=6; hPIEZO1: 1x, n=174/1919; N=18; 4x, n=346/667; N=4; ****P< 0.0001). Time to peak andT50 values of mPIEZO1 (C-D) and hPIEZO1 cells (H-I) recorded from 1x (dark blue) and 4x (purple)
chips (mPIEZO1: 1x, n=88/1440; N=9; 4x, n=506/843; N=5; *P=0.0211, ****P < 0.0001; hPIEZO1: 1x,n=103/1471; N=9; 4x, n=324/667; N=4; * P=0.0375, ****P < 0.0001). Percentage of mPIEZO1 (E) andhPIEZO1 (J) cells responding to activation by M-Stim at 110µl/s pipetting flow using 1x (dark blue) and
4x (purple) chips (mPIEZO1: 1x, N=18; 4x, N=6; hPIEZO1: 1x, N=18; 4x, N=4; ****P < 0.0001). Data areshown as values of individual cells with median, and 95% CI (B, C, D/G, H, I), or as mean ± SEM (E/J),
indicated in black, tested for statistical significance Mann Whitney test and unpaired t-test.

6Summary• Fast pipetting flow (110 µl/s) is a key factor for the mechanicalstimulation (M-Stim) of PIEZO1 channels on the SyncroPatch 384• Activation of hPIEZO1 by M-Stim results in faster signal onset and fastersignal decay compared to mPIEZO1• Using M-Stim with multi-hole (4x) chips increases signal amplitude andnumber of responding wells, and represents a valid tool for the study ofPIEZO1 channels on the SyncroPatch384• M-Stim can be combined with chemical stimulation by e.g. Yoda1 tomaximize the success rate (i.e., number of responding cells)• M-Stim provides a highly parallelized, objectified approach to allow formechanically induced PIEZO1 activation, enabling mutants'
investigations and compound screening, with the ultimate goal ofaccelerating the development of new medical strategies.

Figure 4: Representative mPIEZO1 (A) and hPIEZO1 (F) raw traces of single cells elicited by M-Stim +10µM Yoda1 at 40µl/s (light blue trace), 60µl/s (blue trace) and 110µl/s (dark blue trace) and blocked
by GdCl3 (black trace). Absolute peak current amplitudes of mPIEZO1 (E) and hPIEZO1 cells (I)plotted against pipetting flow (mPIEZO1: 1x, n=465/1353; N=9; 4x, n=685/722; N=6; hPIEZO1: 1x,n=456/1390; N=9; 4x, n=577/604; N=4; ****P < 0.0001). Time to peak and T50 values of mPIEZO1 (C-D)
and hPIEZO1 cells (H-I) plotted against pipetting flow (mPIEZO1: 1x, 40µl/s n=142/1420; 60µl/s
n=242/1388; 110µl/s n=465/1353; N=9; 4x, 110 µl/s n=662/697; N=5; * P=0.0178, ****P < 0.0001; hPIEZO1:1x, 40 µl/s n=104/1425; 60 µl/s n=227/1412; 110 µl/s n=460/1390; N=9; 4x, 110 µl/s n=566/604; *P=0.0094,****P < 0.0001). Percentage of mPIEZO1 (E) and hPIEZO1 (J) cells responding to M-Stim + Yoda1 at
110µl/s pipetting flow recorded from 1x (dark blue) and 4x (purple) chips (mPIEZO1: 1x, N=18; 4x, N=6;hPIEZO1: 1x, N=18; 4x, N=4, ****P < 0.0001). Data are shown as values of individual cells with median,and 95% CI (B, C, D/G, H ,I), or as mean ± SEM (E/J), indicated in black, tested for statisticalsignificance using Kruskal-Wallis test with Dunn’s post-hoc test and unpaired t-test.

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