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Yoda1 analogue (Dooku1) which antagonises Yoda1-evoked activation of Piezo1 and aortic relaxation: Yoda1 antagonist

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Background and purpose: The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small-molecule agonist but the pharmacology of these channels is otherwise limited. Experimental approach: Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+and Tl+measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in human umbilical vein endothelial cells (HUVECs) which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta. Key results: Modification of the pyrazine ring of Yoda1 yielded an analogue which lacked agonist activity but reversibly antagonised Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1-induced Ca2+-entry with IC50s of 1.3 μM (HEK 293 cells) and 1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP-evoked Ca2+elevation or store-operated Ca2+entry in HEK 293 cells or Ca2+entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose-dependent relaxation of aortic rings which was mediated by an endothelium- and nitric oxide-dependent mechanism and which was antagonised by Dooku1 and analogues of Dooku1. Conclusion and implications: Chemical antagonism of Yoda1-evoked Piezo1 channel activity is possible and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.
The 2,6-dichlorophenyl group of Yoda1 is required for activation of Piezo1. (A) Structures of Yoda1 and analogues. Structural variation to Yoda1 is highlighted by the box outline. (B) Western blot of control T-REx and Piezo1 T-REx cells with anti-Piezo1 antibody, confirming Piezo1 expression (predicted size, 286 kDa). (C) Real-time PCR of Piezo1 mRNA levels relative to GAPDH mRNA in T-REx and Piezo1 T-REx cells. Error bars indicate SEM (n = 3). (D and E) FlexStation intracellular Ca 2+ measurement data for T-REx cells (D) and Piezo1 T-REx cells (E) exposed to Yoda1 at the specified concentrations or exposed to the vehicle only (DMSO). (F) (Left) FlexStation intracellular Ca 2+ measurement data for Piezo1 T-REx cells exposed to 10 μM 2e or exposed to vehicle only (DMSO). Error bars indicate SEM (N = 3). (Right) Summary for experiments of the type shown on the left measured between 40-60 s after Yoda1 analogue application, expressed as a % of the 10 μM Yoda1 response. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated in black (n = 5). (G) (Left) FlexStation intracellular Ca 2+ measurement data for Piezo1 T-REx cells exposed to 2 μM Yoda1 after pretreatment with 10 μM 2e or vehicle only (DMSO). Error bars indicate SEM (N = 3). (Right) Summary for experiments of the type shown on the left, as for (F, right) except data are expressed as a % of the Yoda1 response when pretreated with vehicle only (DMSO) (n = 5; 2b, 2c, 2e, 2g, 2h, n = 7; 2a, 2d, 2f).
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RESEARCH PAPER
Yoda1 analogue (Dooku1) which antagonizes
Yoda1-evoked activation of Piezo1 and aortic
relaxation
Correspondence Professor David J Beech, Leeds Institute of Cardiovascular and Metabolic Medicine, LIGHT Building, Clarendon Way,
School of Medicine, University of Leeds, Leeds LS2 9JT, UK. E-mail: d.j.beech@leeds.ac.uk
Dr Richard Foster, School of Chemistry, University of Leeds, Leeds LS2 9JT, UK. E-mail: r.foster@leeds.ac.uk
Received 9 October 2017; Revised 14 February 2018; Accepted 14 February 2018
Elizabeth L Evans
1,
*,KevinCuthbertson
2,
*, Naima Endesh
1,
*, Baptiste Rode
1
,NicolaMBlythe
1
,
Adam J Hyman
1
, Sally J Hall
1
, Hannah J Gaunt
1
,MelanieJLudlow
1
,RichardFoster
2
and David J Beech
1
1
Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds, UK, and
2
School of Chemistry, University of
Leeds, Leeds, UK
*Equal contributors.
BACKGROUND AND PURPOSE
The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small-molecule agonist,
but the pharmacology of these channels is otherwise limited.
EXPERIMENTAL APPROACH
Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca
2+
and Tl
+
measurements were made in HEK 293 or CHO
cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made
from rings of mouse thoracic aorta.
KEY RESULTS
Modication of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1.
The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1-induced Ca
2+
-entry with IC
50
sof1.3μM (HEK 293 cells) and
1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP-evoked Ca
2+
ele-
vation or store-operated Ca
2+
entry in HEK 293 cells or Ca
2+
entry through TRPV4 or TRPC4 channels overexpressed in CHO and
HEK 293 cells. Yoda1 caused dose-dependent relaxation of aortic rings, which was mediated by an endothelium- and NO-
dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1.
CONCLUSION AND IMPLICATIONS
Chemical antagonism of Yoda1-evoked Piezo1 channel activity is possible, and the existence of a specicchemicalinteractionsite
is suggested with distinct binding and efcacy domains.
Abbreviations
SBS, standard bath solution; TRP, transient receptor potential
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
British Journal of
Pharmacology
British Journal of Pharmacology (2018) 175 17441759 1744
DOI:10.1111/bph.14188 © 2018 The Authors. British Journal of Pharmacology
published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.
Introduction
Piezo1 protein is important for mechanical force sensing
and its transduction in higher organisms (Coste et al., 2010;
Ranade et al., 2015; Wu et al., 2016). It assembles as a trimer
with a propeller-like structure around a central ion pore,
which is permeable to the cations Na
+
,K
+
and Ca
2+
(Coste
et al., 2012; 2015; Ge et al., 2015; Guo and MacKinnon,
2017; Saotome et al., 2017; Wu et al., 2017; Zhao et al.,
2018). Mechanical forces that include membrane tension
and laminar owareabletoactivatethechannel(Coste
et al., 2010; Li et al., 2014; Lewis and Grandl, 2015; Syeda
et al., 2016).
Roles of Piezo1 have been identied in embryonic vascu-
lar maturation, BP regulation, physical performance,
hypertension-dependent arterial structural remodelling,
urinary osmoregulation, epithelial homeostasis and axonal
growth (Li et al., 2014; Ranade et al., 2014; Cahalan et al.,
2015; Retailleau et al., 2015; Koser et al., 2016; Martins
et al., 2016; Gudipaty et al., 2017; Rode et al., 2017). In
addition, pathological signicance of Piezo1 has been
suggested in humans. Gain of function mutations have been
linked to a form of haemolytic anaemia (hereditary
stomatocytosis), and loss of function mutations have been
linked to autosomal recessive congenital lymphatic dyspla-
sia (Zarychanski et al., 2012; Albuisson et al., 2013; Andolfo
et al., 2013; Bae et al., 2013; Fotiou et al., 2015; Lukacs
et al., 2015).
Piezo1 pharmacology is in its infancy. Inhibitors of the
channel are limited to generic inhibitors of the ion pore
(Gd
3+
and ruthenium red)andthespidertoxinGsMTx4,
which inhibits a range of mechanosensitive ion channels
and may act indirectly via the lipid bilayer (Drew et al.,
2002; Suchyna et al., 2004; Bowman et al., 2007; Bae et al.,
2011). The rst chemical activator of the channel, Yoda1,
was discovered in 2015 through high-throughput screening
(Syeda et al., 2015). Yoda1 is a useful research tool, not faith-
fully mimicking mechanical stimulation of the channels but
facilitating study of Piezo1 channels from a practical
perspective without the need for mechanical stimulation
and importantly lacking effect on Piezo2 channels
(Cahalan et al., 2015; Lukacs et al., 2015; Wang et al., 2016;
Rode et al., 2017).
As a step towards improved understanding of Piezo1 and
the development of more and better Piezo1 modulators, in-
creased knowledge on the structureactivity relationship for
Yoda1 activation of Piezo1 would be helpful. Here, we ad-
dressed this knowledge gap by synthesizing Yoda1 analogues
and testing them against the Piezo1 channel, other channels
and vascular contractile function.
Methods
Piezo1 tetracycline-inducible HEK 293 cell line
HEK T-REx
cells, which overexpress Piezo1 upon induction
with tetracycline, were made as described in Rode et al.
(2017). Expression was induced by treating the cells for 24 h
with 10 ng·mL
1
tetracycline (Sigma) and analysed by quan-
titative RT-PCR and Western blots.
Cell culture
HEK 293 cells stably expressing tetracycline-regulated human
Piezo1 were utilized as described above. HEK 293 cells stably
expressing tetracycline-regulated human TRPC4 have been
described previously (Akbulut et al., 2015). For the
TRPC4-expressing cells, selection was achieved by including
400 μg·mL
1
zeocin and 5 μg·mL
1
blasticidininthecell
medium. To induce expression, cells were incubated with
1μg·mL
1
tetracycline for 24 h prior to experiments. All
HEK 293 cells were maintained in DMEM (Invitrogen, Paisley,
UK) supplemented with 10% FCS (Sigma-Aldrich) and 1%
penicillin/streptomycin (Sigma-Aldrich).
HUVECs purchased from Lonza were maintained in En-
dothelial Cell Basal Medium. This media was supplemented
with a bullet kit (Cell Media and Bullet Kit, Lonza) contain-
ing the growth factors: 10 ng·mL
1
VEGF, 5 ng·mL
1
hu-
man basic FGF, 1 μg·mL
1
hydrocortisone, 50 ng·mL
1
gentamicin, 50 ng·mL
1
amphotericin B and 10 μg·mL
1
heparin, in addition to 2% FCS (Sigma). HUVECs were
passaged 26times.
CHO K1 cells stably expressing human TRPV4 were
maintained in Hams F12 (ThermoFisher Scientic) in the
presence of 1 mg·mL
1
G418 (Sigma-Aldrich).
All cells were grown at 37°C and in 5% CO
2
in a humidi-
ed incubator.
RT-PCR
Total RNA was extracted using TRI reagent (Sigma-Aldrich).
Five hundred nanograms of total RNA was used for reverse
transcription using the Reverse Transcription System
(Promega). Real-time PCR was conducted using an Applied
Biosystems 7500 Real-Time PCR system with intron spanning
primers and Taqman probe for human Fam38A (Piezo1)
(Hs00207230_m1) and GAPDH (Hs99999905_m1) (Applied
Biosystems).
Western blot for Piezo1 protein
Cells were harvested in lysis buffer [10 mM Tris (pH 7.4),
150 mM NaCl, 0.5 mM EDTA and 0.5% Nonidet P40
substitute] containing protease and phosphatase inhibitor
cocktails (Sigma). Equal amounts of protein were loaded
onto a 420% gradient gel (BioRad) and resolved by
electrophoresis. Samples were transferred to PVDF
membranes and labelled overnight with BEEC-4 (1:1000,
Cambridge Biosciences). HRP-donkey anti-rabbit secondary
antibody (Jackson ImmunoResearch Laboratories) and
SuperSignal Femto detection reagents (Pierce) were used
for visualization.
Intracellular Ca
2+
measurements
HEK 293 and CHO cells were plated in poly-d-lysine coated
96-well plates (Corning, NY,USA)andHUVECsinclear96-
well plates (Corning, NY, USA) at a conuence of 90%, 24 h
before experimentation. Cells were incubated with 2 μM
fura-2-AM (Molecular Probes
)or4μMuo-4-AM (for TRPV4
expressing CHO cells), in the presence of 0.01% pluronic acid
(Thermo Fisher Scientic) in standard bath solution (SBS) for
1 h at 37°C. For recordings with uo-4, 2.5 mM probenecid
(Sigma Aldrich) was included in the SBS throughout the
experiment. Cells were washed with SBS for 30 min at room
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1745
temperature. If inhibitors were being tested, these were added
at this time, immediately following an SBS wash and main-
tained during the rest of the experiment. Measurements were
made at room temperature on a 96-well uorescence plate
reader (FlexStation, Molecular Devices, Sunnyvale, CA, USA)
controlled by Softmax Pro software v5.4.5. For recordings
using fura-2, the change (Δ) in intracellular calcium was indi-
cated as the ratio of fura-2 emission (510 nm) intensities for
340 and 380 nm excitation. For recordings using uo-4, the
dye was excited at 485 nm and emitted light collected at
525 nm, and measurements are shown as absolute uores-
cence in arbitrary units. The SBS contained (mM): 130 NaCl,
5 KCl, 8 D-glucose, 10 HEPES, 1.2 MgCl
2
,1.5CaCl
2
and the
pH was titrated to 7.4 with NaOH. For the Ca
2+
add-back ex-
periments, Ca
2+
free SBS was used (without CaCl
2
), and Ca
2+
add-back was 0.3 mM. For the washout experiments, inhibi-
tors were washed 3 times with SBS immediately prior to
recording.
FluxOR
intracellular Tl
+
(thallium ion)
measurements
Induced (Tet+) and non-induced (Tet) Piezo1 HEK 293 cells
were plated in poly-d-lysine coated 96-well plates (Corning,
NY, USA) and HUVECs in clear 96-well plates (Corning, NY,
USA) at a conuence of 90%, 24 h before experimentation.
Cells were loaded with FluxOR dye for 1 h at room tempera-
ture, before being transferred to assay buffer for 20 min. If
inhibitors were being tested, these were added at this time
and maintained throughout the experiment. Cells were
stimulated with a Tl
+
-containing K
+
-free solution according
to the manufacturers instructions (Molecular Probes).
Measurements were made at room temperature on a 96-well
uorescence plate reader (FlexStation, Molecular Devices,
Sunnyvale, CA, USA) controlled by Softmax Pro software
v5.4.5. FluxOR was excited at 485 nm, emitted light collected
at 520 nm, and measurements were expressed as a ratio
increase over baseline (F/F
0
).
Chemical synthesis of Yoda1 analogues
Analogues of Yoda1 were synthesized using three general
synthetic approaches: 11 compounds [2a-2 k] were synthe-
sized using a one-step procedure (Supporting Information
Figure S1), compounds 7a and 7b using a four-step procedure
(Supporting Information Figure S2) and compound 11 using
a separate four-step procedure (Supporting Information-
Figure S3). All chemicals synthesized were puried by
column chromatography or trituration and determined as
>97% pure by
1
H NMR (proton NMR) and
13
CNMR(car-
bon-13 NMR). Synthetic and analytical details are reported
in the Supporting Information.
Animals
Twelve to sixteen week-old, wild-type male C57BL/6 mice
were used for experiments. All mice were housed in GM500
individually ventilated cages (Animal Care Systems) at 21°C,
5070% humidity and with a 12 h alternating light/dark
cycle. They had ad libitum access to RM1 diet (SpecialDiet
Services, Witham, UK) with bedding from PureoCell
(Datesand, Manchester, UK). All animal experiments were
authorized by the University of Leeds Animal Ethics
Committee and the UK Home Ofce. Animal studies are re-
ported in compliance with the ARRIVE guidelines (Kilkenny
et al., 2010; McGrath and Lilley, 2015).
Aorta contraction studies
Thewiremyographtechniqueusingvesselsfrommiceis
regarded as a useful model for studying vascular reactivity
(Outzen et al., 2015). Animals were killed by CO
2
inhalation,
according to Schedule 1 procedure approved by the UK Home
Ofce. Thoracic aorta was dissected out and immediately
placed into ice-cold Krebs solution (125 mM NaCl, 3.8 mM
KCl, 1.2 mM CaCl
2
,25mMNaHCO
3
,1.2mMKH
2
PO
4
,
1.5 mM MgSO
4
,0.02mMEDTAand8mMD-glucose,
pH 7.4). Connective tissue and fat were carefully removed un-
der a dissection microscope. Segments, 1 mm long, were
mounted in an isometric wire myograph system (Multi Wire
Myograph System, 620 M, Danish Myo Technology) with
two 40 μm diameter stainless steel wires, bathed in Krebs
solution at 37°C and bubbled with 95% O
2
,5%CO
2.
The seg-
ment was then stretched stepwise to its optimum resting ten-
sion to a 90% equivalent transmural pressure of 100 mmHg
and equilibrated for 1 h prior to experiments. The stretch
was approximately equal to that expected at diastolic BP
(Rode et al., 2017).
Data and statistical analysis
The data and statistical analysis comply with the recom-
mendations on experimental design and analysis in phar-
macology (Curtis et al., 2015). OriginPro 2015 (OriginLab,
Northampton, MA, USA) was used for all data analysis.
Averaged data are presented as mean ± SEM, where nrepre-
sents the number of independent experiments for a given
result and Nindicates the total number of replicates within
the independent experiments. Technical replicates were
used to improve the condence in data from independent
experiments. In order to compare the pharmacological
activity of Yoda1 analogues, data were normalized to the
response of Yoda1 (agonist experiments) or the response
of Yoda1 following pretreatment with vehicle only (inhibi-
tor experiments). Data subjected to statistical analysis
contained at least ve independent experiments (n). For
comparisons between two sets of data, Studentst-tests were
used. For multiple comparisons, one-way ANOVA was used
with Tukeyspost hoc test. P<0.05 was deemed signicant.
For IC
50
determination, data were normalized to the vehicle
controls (DMSO), and curves were tted using the Hill1
(Origin Pro 2015) equation. The analogues were novel,
and so, their initial testing occurred without knowledge of
what effects might occur. Later in the study, analogues were
blinded for aorta contraction experiments and used in
random order. Randomization and blinding were not other-
wise used.
Materials
Unless stated otherwise, all commercially available chemicals
were purchased from Sigma-Aldrich. Stocks of chemicals were
reconstituted in DMSO and stored at 20°C unless stated oth-
erwise. Fura-2-AM and uo-4-AM (Molecular Probes) were
dissolved at 1 mM. Pluronic acid F-127 was stored at 10%
w
.
v
-1
in DMSO at room temperature. Probenecid was freshly
prepared in 0.5 M NaOH and diluted 1:200 in SBS to give a
E L Evans et al.
1746 British Journal of Pharmacology (2018) 175 17441759
working concentration of 2.5 mM. Yoda1 (Tocris) was stored
at 10 mM. All Yoda1 analogues were synthesized and puried
(for more information, see Supporting Information) and pre-
pared as 10 mM stock solutions. Stock solutions were diluted
1:500 in the recording solution to give a nal working con-
centration of 0.02% DMSO. Thapsigargin and 4α-
phorbol 12, 13-didecanoate were stored as 5 and 10 mM
stocks respectively. (-)-Englerin A was prepared as a
10 mM stock solution and stored at 80°C. In experiments,
(-)-Englerin A was used in SBS containing 0.01% pluronic acid
as a dispersing agent to minimize aggregation of compound.
Phenylephrine was stored at 100 mM in an aqueous solu-
tion. ATP wasstoredat10mMinanaqueousstocksolution.
U46619 was stored as a 10 mM stock in water. SIN-1 was
stored as a 20 mM stock. BEEC-4 anti-Piezo1 antiserum, di-
luted 1:1000 for experiments, was generated by Cambridge
Biosciences in rabbits by presentation of the Piezo1 peptide
DLAKGGTVEYANEKHMLALA.
Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked
to corresponding entries in http://www.guidetophar-
macology.org, the common portal for data from the
IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al.,
2018), and are permanently archived in the Concise
Guide to PHARMACOLOGY 2017/2018 (Alexander et al.,
2017a,b).
Results
2,6-Dichlorophenyl ring of Yoda1 is important
for Piezo1 activity
We synthesized a series of Yoda1 analogues, focusing on sim-
ple modications to the 2,6-dichlorophenyl ring (Figure 1A).
To reliably study the effects of Yoda1 analogues on
overexpressed Piezo1 channels, we stably incorporated
tetracycline-inducible human Piezo1 expression in HEK
293 T-REx
cells. These cells, hereby referred to as Piezo1
T-REx cells, showed Piezo1 expression after tetracycline
induction but not without induction (Figure 1B, C) and
displayed dose-dependent Ca
2+
entry in response to
Yoda1, in comparison with normal HEK 293 T-REx
cells
(without Piezo1 incorporation) that showed no response
(Figure 1D, E). The Yoda1 analogues were screened at
10 μM for their ability to cause Ca
2+
entry in these Piezo1
T-REx cells and compared with the Ca
2+
entry caused by
thesameconcentrationofYoda1(Figure1F).Allofthe
structural changes caused Piezo1 activation to be lost or
mostly lost, with all compounds showing less than 30% ac-
tivation compared with Yoda1 (Figure 1F).
The analogues were also screened for their ability to
inhibit the Yoda1 response (Figure 1G). Each analogue was
pre-incubated with the cells for 30 min at 10 μM, prior to
the application of 2 μM Yoda1 in the continued presence of
the analogue. Pre-incubation with these analogues did not
affect the Ca
2+
entryevokedbyYoda1,apartfrom2g which
caused inhibition. These data suggest that the 2,6-
dichlorophenyl moiety of Yoda1 is essential for interacting
with the Piezo1 channel. Only analogue 2g had any effect,
showing a slight inhibitory effect but little agonist effect; it
is chemically similar to Yoda1 but with one uorine replacing
one chlorine.
Identification of a Yoda1 analogue which
antagonizes Yoda1
To further investigate the structureactivity relationship of
Yoda1, we synthesized analogues of the pyrazine group
(Figure 2A). Similarly, these analogues were tested at 10 μM
for their ability to cause Ca
2+
entry in Piezo1 T-REx cells, com-
pared with Yoda1 (Figure 2B, C). Modication to the pyrazine
ring signicantly reduced activity in comparison with Yoda1,
but analogue 7a reached 50% of Yoda1 activity (Figure 2B, C).
We then synthesized analogues of the thiadiazole group
(Figure 2D) and tested these in the same manner (Figure 2E, F).
Analogues containing an oxadiazole in place of a thiadiazole
were also less active, but analogue 11, the most similar in
structure to Yoda1, showed ~70% activity (Figure 2E, F).
These data suggest that the ability of Yoda1 to activate
Piezo1 channels is dependent on very specicstructuralre-
quirements but that changes to the pyrazine and thiadiazole
groups can be tolerated.
To investigate whether these analogues could inhibit
Yoda1 activity, we pre-incubated cells with analogues and
then tested Yoda1 (Figure 3AG). The Yoda1 response was re-
duced by all analogues (Figure 3G). Analogues 2i (Figure 3A),
2j (Figure 3B), 7a (Figure 3D), 7b (Figure 3E) and 11 (Figure3F)
also had agonist activity, as shown by the elevated baseline
Ca
2+
signal compared with vehicle (DMSO) control. In con-
trast, analogue 2k (Figure 3C) inhibited the Yoda1 response
without changing the baseline and so lacked agonist activity
(Figure 3C). Analogue 2k was found to cause concentration-
dependent inhibition of Yoda1-induced Ca
2+
entry with an
IC
50
value of 1.30 μM (Figure 3H). Inhibition was incom-
plete at 10 μM, but higher concentrations of 2k were not in-
vestigated because of solubility limitations. Recovery from
the inhibitory effect of 2k occurred after its washout
(Figure 3I). The inhibitory effect of 2k was not signicantly
differentat37°Ccomparedwithroomtemperature(Figure3
J, K). The data suggest that 2k is an antagonist of Yoda1 that
lacks agonist capability. We named 2k, Dooku1.
Dooku1 (analogue 2k) has selectivity for Piezo1
Pretreatment with 10 μM Dooku1 had no effect on endoge-
nous Ca
2+
release in native HEK 293 cells in response to
20 μM ATP (Figure 4A). Dooku1 (10 μM) had no effect on
store-operated Ca
2+
entry in HEK 293 cells: the Ca
2+
addback
response after intracellular Ca
2+
store depletion by 2 μM
thapsigargin (Figure 4B). Dooku1 (10 μM) had no effect on
Ca
2+
entry through TRPV4 channels overexpressed in CHO
cells and activated by 4αPDD (Figure 4C) or on Ca
2+
entry
through TRPC4 channels overexpressed in T-REx
HEK
293 cells and activated by 100 nM (-)-Englerin A (EA)
(Figure 4D). The data suggest selectivity of Dooku1 for
Piezo1 channels.
Dooku1 does not inhibit constitutive Piezo1
activity
To investigate whether the effect of Dooku1 depends on
Yoda1, we took advantage of constitutive Piezo1 channel
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1747
activity observed in the Piezo1 T-REx cells (Rode et al., 2017).
The activity can be detected using an intracellular thallium
(Tl
+
)sensitiveFluxOR
indicator dye whereby Tl
+
inux acts
as a surrogate for Na
+
inux (Rode et al., 2017). Cells were
maintained in a Tl
+
free solution until 2 μMTl
+
was added ex-
tracellularly 30 s into the recording, and the resulting eleva-
tion of intracellular Tl
+
was detected. To ensure that
constitutive Piezo1 channel activity was being represented
in this assay, we compared the rate of Tl
+
entry in
tetracycline-induced (Tet+) Piezo1 overexpressing cells to
control cells to which tetracycline was not added (Tet)
(Figure 5A, B). The initial rate of Tl
+
entry in the Tet + cells
was nearly double that of control Tetcells (Figure 5A, B).
Pretreatment with Dooku1 did not reduce constitutive
Piezo1 channel activity as shown by comparing the DMSO
and Dooku1 DMSO data (Figure 5C, D). Yoda1 increased the
rate of Tl
+
entry by ~2.5-fold, and this effect was inhibited
by 10 μM Dooku1 as shown by comparing the Yoda1 and
Dooku1 Yoda1 data (Figure 5C, D). These data suggest that
Dooku1 has no effect on constitutive Piezo1 channel activity
and therefore that its effect depends on the presence of
Yoda1.
Figure 1
The 2,6-dichlorophenyl group of Yoda1 is required for activation of Piezo1. (A) Structures of Yoda1 and analogues. Structural variation to Yoda1 is
highlighted by the box outline. (B) Western blot of control T-REx and Piezo1 T-REx cells with anti-Piezo1 antibody, conrming Piezo1 expression
(predicted size, 286 kDa). (C) Real-time PCR of Piezo1 mRNA levels relative to GAPDH mRNA in T-REx and Piezo1 T-REx cells. Error bars indicate
SEM (n= 3). (D and E) FlexStation intracellular Ca
2+
measurement data for T-REx cells (D) and Piezo1 T-REx cells (E) exposed to Yoda1 at the spec-
ied concentrations or exposed to the vehicle only (DMSO). (F) (Left) FlexStation intracellular Ca
2+
measurement data for Piezo1 T-REx cells ex-
posed to 10 μM2e or exposed to vehicle only (DMSO). Error bars indicate SEM (N= 3). (Right) Summary for experiments of the type shown on the
left measured between 4060 s after Yoda1 analogue application, expressed as a % of the 10 μM Yoda1 response. Each data point represents a
value from an independent experiment with mean values and error bars representing SEM indicated in black (n= 5). (G) (Left) FlexStation intra-
cellular Ca
2+
measurement data for Piezo1 T-REx cells exposed to 2 μM Yoda1 after pretreatment with 10 μM2e or vehicle only (DMSO). Error
bars indicate SEM (N= 3). (Right) Summary for experiments of the type shown on the left, as for (F, right) except data are expressed as a % of
the Yoda1 response when pretreated with vehicle only (DMSO) (n=5;2b,2c,2e,2g,2h,n=7;2a,2d,2f).
E L Evans et al.
1748 British Journal of Pharmacology (2018) 175 17441759
Dooku1 inhibits endogenous Yoda1-activated
channels
The above studies were on overexpressed Piezo1 channels. To
investigate the relevance to endogenous Piezo1 channels, we
studied HUVECs that robustly express endogenous Piezo1
channels (Li et al., 2014) and display a Piezo1-dependent
Yoda1 response (Rode et al., 2017). Similar to observations
in Piezo1 T-REx cells (Figure 3C), Dooku1 did not evoke
Ca
2+
entry (Figure 6A). Dooku1 was however able to inhibit
the Yoda1 response in HUVECs (Figures 6B, C). Dooku1 had
a concentration-dependent inhibitory effect against Yoda1-
induced Ca
2+
entry in HUVECs, acting with an IC
50
of
1.49 μM (Figure 6D), which was comparable with the value
in Piezo1 T-REx cells even though its maximum effect was less
(Figure 3H). These data suggest that Dooku1 is also an antag-
onist of Yoda1-induced Piezo1 channels in endothelial cells.
To investigate the reason for reduced Dooku1 effect against
the endogenous Yoda1-activated channel, we compared the
concentration-effect curves of Yoda1 in HUVECs (Figure 6E)
and Piezo1 T-REx cells (Figure 6F). Yoda1 had increased
potencyinHUVECswithanEC
50
of 0.23 μM, compared with
2.51 μM in Piezo1 T-REx cells, suggesting that greater Yoda1
potencyinHUVECsmayexplainthesmallereffectofDooku1
in HUVECs.
Yoda1 causes endothelium-dependent and NO-
dependent relaxation of aorta
To investigate physiological responses, we made isometric
tension recordings from isolated murine thoracic aorta rings.
Yoda1 had no effect in the absence of phenylephrine (PE),
whichisanagonistofα
1
-adrenoreceptors (Figure 7A). Rings
contracted in response to PE (Figure 7B) and Yoda1 caused
concentration-dependent relaxation following this pre-
contraction, with an estimated EC
50
of 2.3 μM(Figure7B).
Endothelium-denudation abolished the Yoda1 response but
didnotaffectthePEresponse(Figure7C,D).Responseto
ACh was a positive control for functional endothelium, and
this response was present in endothelium-intact rings but
Figure 2
Changes to the pyrazine ring or replacing the thiadiazole with an oxadiazole give rise to less active analogues. (A) Structures of Yoda1 and
analogues with changes to the pyrazine ring. Structural variation to Yoda1 is highlighted by the box outline. (B) FlexStation intracellular Ca
2+
measurement data for Piezo1 T-REx cells exposed to 10 μM7a or exposed to vehicle only (DMSO). Error bars indicate SEM (N= 3). (C) Summary
for experiments of the type shown in (B) measured between 4060 s after Yoda1 analogue application, expressed as a % of the 10 μMYoda1
response. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated in
black (n= 5). (D) Structures of Yoda1 analogues with an oxadiazole. Structural variation to Yoda1 is highlighted by the box outline. (E) FlexStation
intracellular Ca
2+
measurement data for Piezo1 T-REx cells exposed to 10 μM2j or exposed to vehicle only (DMSO). Error bars indicate SEM
(N= 3). (F) Summary for experiments of the type shown in (E), as for (C).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1749
Figure 3
Yoda1 analogues are able to inhibit Yoda1-induced Piezo1 activity. (AF) FlexStation intracellular Ca
2+
measurement data for Piezo1 T-REx cells
exposed to 2 μM Yoda1 after pretreatment with 10 μM2i (A), 2j (B), 2k (C), 7a (D), 7b (E), 11 (F) or vehicle only (DMSO). Error bars indicate
SEM (N= 3). (G) Summary for experiments of the type shown in (AF) measured between 4060 s after Yoda1 analogue application, expressed
as a % of the Yoda1 response when pretreated with vehicle only (DMSO). Each data point represents a value from an independent experiment
with mean values and error bars representing SEM indicated in black (n= 5). (H) Mean data for the type of experiment shown in (C) with cells
pretreated with indicated concentrations of 2k. Expressed as a % of the Yoda1 response when pretreated with vehicle only (DMSO). The tted
curve is the Hill equation with IC
50
1.30 μM(n= 5). (I) Summary of intracellular Ca
2+
measurement data (as for G) for Tet + Piezo1 T-REx cells
exposed to 2 μM Yoda1, following pretreatment with 10 μM2k or vehicle only (DMSO); 2k was washed out before the recording (n= 5). (J)
As for (C) but conducted at 37°C. (K) Summary for experiments of the type shown in (J) (n=5).
E L Evans et al.
1750 British Journal of Pharmacology (2018) 175 17441759
absent in endothelium-denuded rings (Figure 7C). The data
suggest that Yoda1 causes endothelium-dependent relaxa-
tion in mouse thoracic aorta.
To determine whether the relaxation caused by Yoda1
was dependent on NOS, we exposed rings to NOS inhibitor,
Nω-nitro-L-arginine methyl ester (L-NAME). L-NAME
Figure 5
Dooku1 does not affect Piezo1 constitutive activity (A) Intracellular Tl
+
measurement data using FluxOR for Tet + Piezo1 T-REx cells or control Tet
cells exposed to extracellular Tl
+
. The FluxOR measurements are displayed as the uorescence intensity (F) divided by the initial uorescence in-
tensity (F
0
). Error bars indicate SEM (N= 3). (B) Summary for experiments of the type shown in (A) measured between 030 s after Tl
+
application,
normalized to rate of change of F in the Tetresponse. Each data point represents a value from an independent experiment with mean values and
error bars representing SEM indicated in black (n= 5). (C) Intracellular Tl
+
measurement data for Tet + Piezo1 T-REx cells exposed to extracellular
Tl
+
and 5 μM Yoda1 or vehicle (DMSO), following pretreatment with 10 μM Dooku1 or vehicle only (DMSO). Error bars indicate SEM (N= 3). (D)
Summary for experiments of the type shown in (C), as for (B) except data are normalized to the rate of change of the vehicle only (DMSO) control
condition (n=5).
Figure 4
Selectivity of Dooku1. Ca
2+
indicator dyes were fura-2 (A, B, D) or uo-4 (C). Experiments conducted in native HEK 293 cells (A, B), CHO cells over-
expressing TRPV4 (C) or HEK 293 cells overexpressing TRPC4 (D). Intracellular Ca
2+
measurement data for cells exposed to 20 μM ATP (A), 0.3 mM
Ca
2+
addback (B), 5 μM4α-phorbol 12,13-didecanoate (4α-PDD) (C) or 100 nM (-)-Englerin A (EA) (D) following pretreatment with DMSO or
10 μM Dooku1 (left). Error bars indicate SEM (N= 3). Summary for experiments of the type shown on the left measured between 1030 s (A),
6090 s (B), 220240s(C)or2060 s (D) after treatment application and normalized to the peak amplitude values for the vehicle only (DMSO)
pretreatment condition (right). Each data point represents a value from an independent experiment with mean values and error bars representing
SEM indicated in black (n=5).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1751
prevented Yoda1-induced and ACh-induced relaxation
(Figure 7E, F). The data suggest that Yoda1 causes
endothelium-dependent relaxation in mouse thoracic aorta
by stimulating NO production via the endothelium.
Dooku1 inhibits Yoda1-induced relaxation of
aorta
To determine if Dooku1 inhibits relaxation caused by Yoda1,
aortic rings were pre-incubated with 10 μMDooku1for
20 min. Dooku1 strongly suppressed the Yoda1-induced
relaxation (Figure 8AC). To characterize this phenomenon
in more detail, we tested four further Yoda1 analogues in
the aorta assay. The selected analogues showed various
abilities to inhibit Yoda1 responses in Piezo1 T-REx cells:
analogues 2e (no activation and no inhibition) (Figure 1),
2g (slight activation and slight inhibition) (Figure 1), 7b
(slight activation and partial inhibition) (Figures 2 and 3)
and 11 (slight activation and partial inhibition) (Figures 2
and 3). Analogue 2e had no effect (Figure 8DF). 2g,7b and
Figure 6
Dooku1 is effective against the endogenous Piezo1 channel. (A) Intracellular Ca
2+
in HUVECs after exposure to 10 μM Dooku1 or vehicle only
(DMSO). Error bars indicate SEM (N= 3). (B) Intracellular Ca
2+
in HUVECs after exposure to 2 μM Yoda1 after pretreatment with 10 μM Dooku1
or vehicle only (DMSO). Error bars indicate SEM (N= 3). (C) Summary for experiments of the type shown in (B) measured 4060 s after Yoda1
application and normalized to peak amplitudes for the vehicle only group. Each data point represents a value from an independent experiment
with mean values and error bars representing SEM indicated in black (n= 7). (D) Mean data for the type of experiment shown in (B) with cells
pretreated with indicated concentrations of Dooku1. Data are expressed as a % of the Yoda1 response when pretreated with vehicle only (DMSO).
The tted curve is the Hill equation with IC
50
1.49 μM(n= 5). (E, F) Mean intracellular Ca
2+
for HUVECs (E) or Piezo1 T-REx cells (F) exposed to the
indicated concentrations of Yoda1. The tted curve is the Hill equation with EC
50
of 0.23 μM(E)and2.51μM(F)(n=3).
E L Evans et al.
1752 British Journal of Pharmacology (2018) 175 17441759
11 in contrast suppressed the Yoda1-induced relaxation
(Figure 8GK). Moreover, the ability of these analogues to
inhibit Yoda1-induced relaxation correlated with inhibition
of Yoda1-induced Ca
2+
entry (Figure 8L). The data suggest
strong efcacy of Dooku1 as an inhibitor of Yoda1-induced
aortic relaxation that is mediated through disruption of
Yoda1-induced Piezo1 channel activity.
Dooku1 is selective for Yoda1-induced
relaxation but partially inhibits agonist
contractile responses
AnalysisofthePEresponseinthepresenceofDooku1
revealed signicant inhibition without effect on baseline
tension (Figure 9A, B). To determine whether Dooku1s
inhibition of PE-induced contraction was specictothis
contractile agent, we also tested the effect of Dooku1
against contraction induced by U46619, a Tx A
2
mimetic.
Aortic rings were pre-contracted with 0.1 μM U46619
(Figure 9C, D). Addition of Dooku1 caused partial relaxation
(Figure 9D, E). In contrast, Dooku1 had no effect on relaxa-
tion evoked by ACh (1 μM) or the NO donor SIN-1 (10 μM)
(Figure9F,G).InvestigationofthePEresponseinthe
presence of the other four Yoda1 analogues revealed no
inhibitory effect (Figure 10). The data suggest that Dooku1
selectively inhibits Yoda1-induced relaxation but also
partially inhibits receptor-mediated agonist responses via
unknown mechanisms.
Discussion and conclusions
This study has provided insight into the structureactivity
relationships for Piezo1 channel activation by Yoda1 with
the goal of generating new tools for investigating Piezo1
channel function. Through this research, we have identied
and named Dooku1, an inhibitor of Yoda1-induced Piezo1
channel activity that strongly inhibits Yoda1-induced
Figure 7
Yoda1-induced relaxation in mouse thoracic aorta is endothelium- and NO-dependent. (A) Isometric tension recording from aorta exposed to the
indicated concentrations of Yoda1. (B) (left) As for (A) but following pre-constriction with 0.3 μM phenylephrine (PE). (Right) Mean data for ex-
periments of the type shown on the left expressed as % relaxation. The tted curve is the Hill equation with EC
50
of 2.3 μM(n= 5). (C) Isometric
tension recording of aorta pre-constricted with PE and exposed to 5 μM Yoda1 (left) or 5 μM ACh control (middle and right) with the endothelial
layer removed (left and middle) or intact (right). (D) Summary data for experiments of the type shown in (B and C, left) expressed as % relaxation
evoked by Yoda1 (left) or the response to PE (right) in the presence (EC+) or absence (EC) of the endothelial cell layer. Each data point represents
a value from an independent experiment with mean values and error bars representing SEM indicated by the black lines (n= 5). (E) As for (C) but
following pre-incubation with 100 μMN
ω-
nitro-L-arginine methyl ester (L-NAME). (F) As for (D) but for experiments of the type shown in (E).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1753
relaxation of aorta. The data suggest that Dooku1 may
compete with Yoda1 at a binding site or act allosterically at
another site to reduce the binding or efcacy of Yoda1.
During the discovery of Yoda1, the 2,6-dichlorophenyl
group of the compound was highlighted as important with
particular reference to the chlorine atoms (Syeda et al.,
Figure 8
Dooku1 inhibits Yoda1-induced dilation in aorta. (AK) Isometric tension data from mouse thoracic aorta with intact endothelium. (A) Pre-con-
stricted with PE and exposed to 5 μM Yoda1. (B) As for (A) but following 30 min pre-incubation with 10 μM Dooku1. (C) Summary data for ex-
periments of the type shown in (A, B) expressed as % relaxation evoked by Yoda1. Each data point represents a value from an independent
experiment with mean values and error bars representing SEM indicated by the black lines (n= 7). (DF) (GI) As for (AC) but following pre-in-
cubation with 10 μM2e (DF) or 7b (GI) (n= 5 on F, I). (J, K) As for (C) but following pre-incubation with 10 μM2g (J) or 11 (K) (n= 5). (L)
Comparison of the mean % inhibition of Yoda1-induced relaxation in mouse thoracic aorta and the mean % inhibition of Yoda1-induced Ca
2+
entry by the ve compounds: 2e,2g,Dooku1,7b and 11.Thepointsaret to a straight line with Pearsons correlation coefcient of 0.78.
E L Evans et al.
1754 British Journal of Pharmacology (2018) 175 17441759
2015). Our ndings support this conclusion and add new
knowledge by demonstrating that small changes to this
group result in complete loss of Piezo1 channel activation.
Removing one of the chlorine atoms [2b] or altering the posi-
tion of the chlorine atom around the ring [2c/2d] abolished
activity. Replacing one or both of the chlorine atoms with
uorine [2a/g] also abolished activity implying that both
chlorine atoms are important for activity and may interact
with Piezo1 in a chlorine specic manner, potentially via a
σ-hole interaction, such as a halogen-pi bond. The
4-methoxyphenyl [2e] and 4-nitrophenyl [2f]analogues
were also inactive. Investigating the inhibitory potential of
the compounds showed that all but 2g,whichisthemost
similar in structure to Yoda1, were ineffective at inhibiting
Yoda1 activity.
Piezo1 channel activation by analogues with modica-
tion to the pyrazine group was less than that of Yoda1, with
the most successful analogue, compound 7a,inwhichthe
pyrazine was replaced with a 3-pyridyl group, exhibiting
50% of the activity of Yoda1. This demonstrates the impor-
tance of the nitrogen atom in the 2-position of the pyrazine
ring, with loss of this nitrogen resulting in a 50% drop of
activity. The remaining two compounds from the series, the
phenyl [2i] and 2-pyrrolyl [7b] analogues, were less active
Figure 9
Specicity of Dooku1 in aorta. All experiments were performed on mouse thoracic aorta with intact endothelium. (A, B) Summary data for exper-
iments of the type shown in Figure 8A, B, expressed as the response to PE (A) or resting tension (B) before and after pre-incubation with 10 μM
Dooku1. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated by
the black lines (n= 7). (C) Aorta were pre-constricted with 0.1 μM U46619 and treated consecutively with DMSO, 1 μMAChand10μMSIN-
1. (D) As for C but pretreated with Dooku1 instead of DMSO. (EG) Summary data for experiments of the type shown in (C, D) expressed as %
of the effect of Dooku1 on the contraction by U46619 (E) or % relaxation evoked by ACh (F) or SIN-1 (G) before and after pre-incubation with
10 μM Dooku1. Each data point represents a value from an independent experiment with mean values and error bars representing SEM indicated
by the black lines (n=5).
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1755
than 7a that suggests that the presence of the nitrogen atom
at the 3-position of the pyridine ring in 7a is also contribut-
ing to Piezo1 activation, supporting our understanding of
the importance of the nitrogen atom at the equivalent posi-
tion on the pyrazine ring of Yoda1 to activity. We next inves-
tigated replacement of the central thiadiazole ring by an
oxadiazole [11]. This change was largely tolerated with the
new compound demonstrating 70% of the activity of Yoda1.
The other two compounds from the series were less active,
although the data for the 2-pyridyl analogue [2j] were inter-
esting in that the partial activity observed for the analogue
suggests that the position of the nitrogen atom on the pyri-
dine contributes to activity, reinforcing the importance of
the equivalent Non the pyrazine ring of Yoda1 to activity.
Investigation into the inhibitory potential of this set of
left-hand and middle ring-modied analogues provided
Figure 10
Lack of effect of other Yoda1 analogues on PE-induced contraction. Summary data for experiments of the type shown in Figure 8 DE, GH
expressed as resting tension (left) or the response to PE (right) following pre-incubation with 10 μM2e (A), 2g (B), 7b (C) and 11 (D). Each data
point represents a value from an independent experiment with mean values and error bars representing SEM indicated by the black lines (n=5).
E L Evans et al.
1756 British Journal of Pharmacology (2018) 175 17441759
compounds with potential promise of being pharmacologi-
cal tools. All of the compounds from the series had the abil-
ity to reduce Ca
2+
entry evoked by Yoda1 by at least 40%,
andasmuchas75%inthecaseof2j. However, most of
these compounds exhibited partial agonist activity. The
most promising compound, 2k (Dooku1) effectively
reduced Yoda1 activity by 60%, without causing any
activation and was a strong inhibitor of the Yoda1 response
in the physiological setting of murine aortic rings. This
shows that the pyrazine ring can be replaced to identify
compounds, which do not activate the channel but do in-
hibit the Yoda1 response. It appears that analogues lacking
the 2,6-dichlorophenyl group do not activate the channels
or inhibit Yoda1 whereas pyrazine-modied analogues show
reduced activation and ability to inhibit Yoda1. Therefore,
the di-chloro group seems to be critical for binding while
the pyrazine group is less important for binding but key
for channel activation.
Currently, the only available inhibitors of Piezo1 activity
are not selective for Piezo1 (Drew et al., 2002; Bae et al.,
2011). Dooku1 is also not perfect as it does not directly block
the channels, but it is a new tool compound that is useful for
Piezo1 characterization studies. It antagonizes the action of
Yoda1 and could facilitate understanding of an important
small-molecule binding site on or near to Piezo1 channels.
Without agonist activity, Dooku1 effectively inhibits Yoda1-
induced Piezo1 activity. It does so without disturbing several
Ca
2+
handling events in the cell or affecting other aortic
relaxing agents. Although these data suggest specicity of
Dooku1 for Piezo1 channels, further studies to address this
point are warranted, especially given the inhibitory effect of
Dooku1 against PE and U46619-induced contractions of aor-
tic rings that might reect a Piezo1 mechanism or some other
unknown effect of Dooku1. It is possible that Dooku1 may be
acting on Piezo1 in smooth muscle cells of the vessel,
partially inhibiting contraction. This assumes that the
channels become activated via a Yoda1-like mechanism
during contraction. Piezo1 was found not be required for
normal myogenic tone (Retailleau et al., 2015), and so, a
non-Piezo1 target of Dooku1 should be considered.
Dooku1 only has activity against Yoda1-induced and not
constitutive Piezo1 channel activity. Such an effect is consis-
tent with Dooku1 acting at the same or a similar site to Yoda1
and thereby occluding access of Yoda1 to its agonist binding
site. The reversibility of Dooku1 is consistent with the
reversibility of Yoda1 (Rocio Servin-Vences et al., 2017). It
would be good to investigate if the Dooku1 effect is consis-
tent with competitive antagonism, but solubility limitations
of the compounds prevented construction of appropriate
concentrationresponse curves. The inability of Dooku1 to
have any effect on constitutive activity suggests that the
mechanism of background channel activity is different to
that of chemical activation with Yoda1.
Dooku1 partially inhibited Yoda1 in HUVECs but
strongly inhibited it in aorta (Figure 6D cf. Figure 8C). We ini-
tially speculated that the difference was due to the higher
temperature of the contraction studies (37°C cf. room
temperature), but the Dooku1 effect was not signicantly
temperature dependent (Figure 3K). An alternative explana-
tion might be that Ca
2+
entry is not directly proportional to
NO production, so that partial inhibition of Yoda-1 induced
Ca
2+
entry is sufcient to inhibit most of the relaxation in-
duced by Yoda1. Another divergence was that Yoda1 was
more potent in HUVECs than Piezo1 T-REx cells, showing a
difference between native and over-expressed Piezo1 chan-
nels (Figure 6E, F). We speculate that this difference reected
a higher basal state of activity of the channels in endothelial
cells, as described previously (Rode et al., 2017), making the
channels more sensitive to Yoda1 because they are better
primed for opening.
In summary, this study has provided important insight
into the structureactivity relationships of Yoda1 and sup-
ported the concept of a specic chemical binding site on or
in close proximity to Piezo1 channels. It has also revealed
the discovery of a useful tool compound, Dooku1, which ef-
fectively antagonizes Yoda1-induced Piezo1 channel activity,
distinguishing it from constitutive Piezo1 channel activity.
The complete role of Piezo1 in vascular biology is still being
established, but the protein may have signicant clinical
interest with emerging roles in genetic disease, BP control,
hypertension-induced arterial remodelling and exercise ca-
pacity (Retailleau et al., 2015; Wang et al., 2016; Rode et al.,
2017). As yet, it is not clear whether activating or inhibiting
this channel may be advantageous, but increasing our
pharmacological knowledge, alongside our physiological
knowledge of Piezo1 will be essential if therapeutic potential
of this protein is to be harnessed in the future. Learning more
about Piezo1 channel interactions with small-molecules
promises to be an important aspect of the overall effort to
understand Piezo1 biology.
Acknowledgements
This work was supported by research grants from the
Wellcome Trust, Medical Research Council and British Heart
Foundation and a studentship from the BBSRC for A.J.H.
and the Libyan Government for N.E.
Author contributions
E.L.E., K.C., N.E., B.R., N.M.B., A.J.H., S.J.H., H.J.G. and M.J.L.
performedexperimentsanddataanalysis.E.L.E.,K.C.,D.J.B.
and R.F. designed the research. D.J.B. and R.F. raised funds
to support the work. E.L.E., K.C., R.F. and D.J.B. co-wrote
the paper.
Conict of interest
The authors declare no conicts of interest.
Declaration of transparency and
scienticrigour
This Declaration acknowledges that this paper adheres to the
principles for transparent reporting and scienticrigourof
preclinical research recommended by funding agencies,
publishers and other organisations engaged with supporting
research.
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1757
References
AkbulutY,GauntHJ,MurakiK,LudlowMJ,AmerMS,BrunsAet al.
(2015). ()-Englerin A is a potent and selective activator of TRPC4
and TRPC5 calcium channels. Angew Chem Int Ed 54: 37873791.
Albuisson J, Murthy SE, Bandell M, Coste B, Louis-Dit-Picard H,
Mathur J et al. (2013). Dehydrated hereditary stomatocytosis linked
to gain-of-function mutations in mechanically activated PIEZO1 ion
channels. Nat Commun 4: 1884.
Alexander SPH, Kelly E, Marrion NV, Peters JA, Faccenda E, Harding
SD et al. (2017a). The Concise Guide to PHARMACOLOGY 2017/18:
Other ion channels. Br J Pharmacol 174: S195S207.
Alexander SPH, Striessnig J, Kelly E, Marrion NV, Peters JA,
Faccenda E et al. (2017b). The Concise Guide to PHARMACOLOGY
2017/18: Voltage-gated ion channels. Br J Pharmacol 174: S160S194.
Andolfo I, Alper SL, De Franceschi L, Auriemma C, Russo R, De Falco L
et al. (2013). Multiple clinical forms of dehydrated hereditary
stomatocytosis arise from mutations in PIEZO1. Blood 121:
39253935.
Bae C, Gnanasambandam R, Nicolai C, Sachs F, Gottlieb PA (2013).
Xerocytosis is caused by mutations that alter the kinetics of the
mechanosensitive channel PIEZO1. Proc Natl Acad Sci 110:
E1162E1168.
Bae C, Sachs F, Gottlieb PA (2011). The mechanosensitive ion
channel Piezo1 is inhibited by the peptide GsMTx4. Biochemistry 50:
62956300.
Bowman CL, Gottlieb PA, Suchyna TM, Murphy YK, Sachs F (2007).
Mechanosensitive ion channels and the peptide inhibitor GsMTx-4:
history, properties, mechanisms and pharmacology. Toxicon 49:
249270.
Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A
(2015). Piezo1 links mechanical forces to red blood cell volume. Elife
4. https://doi.org/10.7554/eLife.07370.
Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ et al.
(2010). Piezo1 and Piezo2 are essential components of distinct
mechanically-activated cation channels. Science (New York, NY) 330:
5560.
Coste B, Murthy SE, Mathur J, Schmidt M, Mechioukhi Y, Delmas P
et al. (2015). Piezo1 ion channel pore properties are dictated by
C-terminal region. Nat Commun 6: 7223.
Coste B, Xiao B, Santos JS, Syeda R, Grandl J, Spencer KS et al. (2012).
Piezo proteins are pore-forming subunits of mechanically activated
channels. Nature 483: 176181.
Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SPA, Giembycz
MA et al. (2015). Experimental design and analysis and their
reporting: new guidance for publication in BJP. Br J Pharmacol 172:
34613471.
Drew LJ, Wood JN, Cesare P (2002). Distinct mechanosensitive
properties of capsaicin-sensitive and -insensitive sensory neurons.
J Neurosci 22: RC228RC228.
Fotiou E, Martin-Almedina S, Simpson MA, Lin S, Gordon K, Brice G
et al. (2015). Novel mutations in PIEZO1 cause an autosomal recessive
generalized lymphatic dysplasia with non-immune hydrops fetalis.
Nat Commun 6: 8085.
Ge J, Li W, Zhao Q, Li N, Chen M, Zhi P et al. (2015). Architecture of
the mammalian mechanosensitive Piezo1 channel. Nature 527:
6469.
Gudipaty SA, Lindblom J, Loftus PD, Redd MJ, Edes K, Davey CF et al.
(2017). Mechanical stretch triggers rapid epithelial cell division
through Piezo1. Nature 543: 118121.
Guo YR, MacKinnon R (2017). Structure-based membrane dome
mechanism for Piezo mechanosensitivity. Elife 6: e33660.
Harding SD, Sharman J, Faccenda E, Southan C, Pawson A, Ireland S
et al. (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018:
updates and expansion to encompass the new Guide to
IMMUNOPHARMACOLOGY. Nucl Acids Res 46: D1091D1106.
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010).
Animal research: reporting in vivo experiments: The ARRIVE
guidelines. Br J Pharmacol 160: 15771579.
Koser DE, Thompson AJ, Foster SK, Dwivedy A, Pillai EK, Sheridan GK
et al. (2016). Mechanosensing is critical for axon growth in the
developing brain. Nat Neurosci 19: 15921598.
Lewis AH, Grandl J (2015). Mechanical sensitivity of Piezo1 ion
channels can be tuned by cellular membrane tension. Elife 4: e12088.
LiJ,HouB,TumovaS,MurakiK,BrunsA,LudlowMJet al. (2014).
Piezo1 integration of vascular architecture with physiological force.
Nature 515: 279282.
Lukacs V, Mathur J, Mao R, Bayrak-Toydemir P, Procter M, Cahalan
SM et al. (2015). Impaired PIEZO1 function in patients with a novel
autosomal recessive congenital lymphatic dysplasia. Nat Commun 6:
8329.
Martins JR, Penton D, Peyronnet R, Arhatte M, Moro C, Picard N et al.
(2016). Piezo1-dependent regulation of urinary osmolarity. Pügers
Arch Eur J Physiol 468: 11971206.
McGrath JC, Lilley E (2015). Implementing guidelines on reporting
research using animals (ARRIVE etc.): new requirements for
publication in BJP. Br J Pharmacol 172: 31893193.
Outzen EM, Zaki M, Abdolalizadeh B, Sams A, Boonen HCM,
Sheykhzade M (2015). Translational value of mechanical and
vasomotor properties of mouse isolated mesenteric resistance-sized
arteries. Pharmacol Res Perspect 3: e00200.
Ranade SS, Qiu Z, Woo S-H, Hur SS, Murthy SE, Cahalan SM et al.
(2014). Piezo1, a mechanically activated ion channel, is required for
vascular development in mice. Proc Natl Acad Sci 111: 1034710352.
Ranade SS, Syeda R, Patapoutian A (2015). Mechanically activated ion
channels. Neuron 87: 1162117 9.
Retailleau K, Duprat F, Arhatte M, Ranade SS, Peyronnet R, Martins JR
et al. (2015). Piezo1 in smooth muscle cells is involved in
hypertension-dependent arterial remodeling. Cell Rep 13:
116 1117 1.
Rocio Servin-Vences M, Moroni M, Lewin GR, Poole K (2017). Direct
measurement of TRPV4 and PIEZO1 activity reveals multiple
mechanotransduction pathways in chondrocytes. Elife 6: e21074.
Rode B, Shi J, Endesh N, Drinkhill MJ, Webster PJ, Lotteau SJ et al.
(2017). Piezo1 channels sense whole body physical activity to reset
cardiovascular homeostasis and enhance performance. Nat Commun
8: 350.
Saotome K, Murthy SE, Kefauver JM, Whitwam T, Patapoutian A,
Ward AB (2017). Structure of the mechanically activated ion channel
Piezo1. Nature 554: 481486.
Suchyna TM, Tape SE, Koeppe RE II, Andersen OS, Sachs F,
Gottlieb PA (2004). Bilayer-dependent inhibition of
mechanosensitive channels by neuroactive peptide enantiomers.
Nature 430: 235240.
E L Evans et al.
1758 British Journal of Pharmacology (2018) 175 17441759
Syeda R, Florendo MN, Cox CD, Kefauver JM, Santos JS, Martinac B
et al. (2016). Piezo1 channels are inherently mechanosensitive. Cell
Rep 17: 17391746.
Syeda R, Xu J, Dubin AE, Coste B, Mathur J, Huynh Tet al. (2015).
Chemical activation of the mechanotransduction channel Piezo1.
Elife : 4, e07369.
Wang S, Chennupati R, Kaur H, Iring A, Wettschureck N, Offermanns
S (2016). Endothelial cation channel PIEZO1 controls blood pressure
by mediating ow-induced ATP release. J Clin Invest 126: 45274536.
Wu J, Goyal R, Grandl J (2016). Localized force application reveals
mechanically sensitive domains of Piezo1. Nat Commun 7: 12939.
Wu J, Lewis AH, Grandl J (2017). Touch, tension, and transduction
the function and regulation of Piezo ion channels. Trends Biochem
Sci 42: 5771.
Zarychanski R, Schulz VP, Houston BL, Maksimova Y, Houston DS,
Smith B et al. (2012). Mutations in the mechanotransduction protein
PIEZO1 are associated with hereditary xerocytosis. Blood 120:
19081915.
Zhao Q, Zhou H, Chi S, Wang Y, Wang J, Geng J et al. (2018). Structure
and mechanogating mechanism of the Piezo1 channel. Nature 554:
487492.
Supporting Information
Additional Supporting Information may be found online in
the supporting information tab for this article.
https://doi.org/10.1111/bph.14188
Figure S1 The general thiol alkylation reaction used to pro-
duce 11 compounds.
Figure S2 General synthetic route towards 7a and 7b. 2,6-
dichlorobenzyl chloride (3) is rst converted to the thiol 4
followed by an SNAr to give 5, which is then brominated to
give 6 ready for a Suzuki cross-coupling to give the desired
products 7a-b.
Figure S3 Synthetic route for 10. 2,6-dichlorobenzyl chlo-
ride (3) is rst converted to the thiol 4 ready for a reaction
with CDI and hydrazine to afford 8. Compound 8 is then uti-
lized in an amide coupling with 9 using EDCI to produce 10
ready for a cyclising-condensation reaction to afford 11.
Yoda1 antagonist
British Journal of Pharmacology (2018) 175 17441759 1759

Supplementary resource (1)

... Due to the different polarity, the binding sites of at an early stage. In a recent publication, the effect of Yoda1 has been reversed by Dooku1, a Yoda1 analogue generated by modifying the pyrazine ring (Evans et al., 2018). The common inhibitor of mechanosensitive cation channels, the GsMTx4 toxin, has shown to have a blocking effect on Piezo1 currents by acting as gating modifier on the closed state of the channel, meaning that greater stress is required to open the channel (Bae et al., 2011). ...
... Difficulties in determining the EC50 for Yoda1 are in agreement with the literature, reporting divergent EC50 values of Yoda1. For instance, an apparent EC50 of 17.1 and 26.6 µM was determined respectively for mouse and human PIEZO1 stably expressed in HEK cells ; while in another HEK cell line overexpressing tetracyclineregulated human Piezo1 (HEK T-REx™) EC50 was found to be 2.51 µM (Evans et al., 2018). ...
... In primary cells, Piezo1 channels respond to Yoda1 at lower micromolar concentrations (Cahalan et al., 2015;Rode et al., 2017;Morley et al., 2018). EC50 values range from 0.23 µM in human umbilical vein endothelial cells (HUVECs) (Evans et al., 2018) to 0.71 μM in human cardiac fibroblasts (Blythe et al., 2019) up to 9 µM in β-cells (Deivasikamani et al., 2019), probably due to tissue-specific expression rates of endogenous Piezo1 channels and different experimental conditions. It is worth mentioning that most of the studies stated above used Transferring the assay to the SyncroPatch 384PE allowed for robust and reliable statistics: 60 % N2A were considered as Yoda1 responders following application of strict quality filters, thus confirming the outcome of preliminary tests done on the Patchliner (48 % success rate). ...
Thesis
Despite the morphological simplicity, the Red Blood Cell (RBC) membrane is endowed with a number of transporters and ion channels, yet not fully characterized and whose biological role is still poorly understood. Most of the techniques used to investigate ion channels are addressed to large populations of cells, thus concealing any putative cell-to-cell variability. The patch clamp technique has proven to be a valid tool for the discovery and characterization of ion channels at a single-cell level. This is of particular relevance for mammalian RBCs, which present a high heterogeneity of conductance not only between different donors but also among cells of the same donor (Kaestner et al., 2004; Minetti et al., 2013). The advent of automated patch clamp allowed to probe an increased number of cells at the same time under identical experimental conditions, thus tackling cell heterogeneity issues. In this thesis, Gárdos and Piezo1 channels were selected as main targets of investigation due to their relevance in RBC-related diseases, i.e. Gárdos channelopathy (Fermo et al., 2017) and hereditary xerocytosis (Zarychanski et al., 2012; Bae et al., 2013). The aim of this work was to develop automated patch clamp assays for characterizing those channels in RBCs. As for Gárdos channels, whole cell recordings reported so far are fragmentary probably due to the low expression of the protein in circulating RBCs (Grygorczyk et al., 1984; Wolff et al., 1988). By increasing the number of cells recorded at the same time, the automated patch clamp technology allowed to identify Gárdos-mediated currents in primary cells with a low-copy number of channels and a large heterogeneity of conductance as RBCs. Piezo1 channels investigations confirmed that application of Yoda1 alone is able to elicit currents sensitive to GdCl3 (non-specific stretch-activated channels inhibitor) but not TRAM-34 (specific Gárdos channel blocker). When transferred to patients carrying a novel PIEZO1 R2110W mutation, the assay revealed that the number of responders and the magnitude of the response to Yoda1 increased in patient compared to control RBCs. This result, combined with structural studies identifying the R2110W residue in a gating sensitive area of the channel, suggested that the novel Piezo1 mutation is gain-of-function (Rotordam et al., 2019). Altogether, this work demonstrates that automated patch clamping provides robust assays to investigate ion channels (Gárdos and Piezo1) in primary cells. The high-throughput technology allowed to tackle issues as response heterogeneity and low expression of the channels, and to characterize a novel channel mutation at a functional level directly from patient cells, without having to express the mutation in a heterologous expression system. This approach may be used to detect other channelopathies not limited to RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing.
... Later, two other agonists, Jedi1 and Jedi2, were discovered [23]. Moreover, Dooku1 was shown to reversibly block Yoda1-evoked activation of Piezo1, leaving unaltered constitutive Piezo1 activity [24]. ...
Article
Full-text available
Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.
... 54 ), the authors reported increased activity of calcineurin and calpain without increased expression of either protein. Stimulation of Piezo1 with Yoda1 produces a tonic increase in the Ca 2+ concentration 54,55 , which is well known to activate calcineurin 43 , but this is not relevant to the high-amplitude Ca 2+ signal produced by aortic constriction and necessary for CaMKII activation 41,42 . Given the differences also in maturity between NRVCMs and adult cardiomyocytes and the absence of data on CaMKII activation in their study of NRVCMs, it is difficult to determine the relevance of the NRVCM data to our findings in adult mice. ...
Article
Full-text available
Pressure overload-induced cardiac hypertrophy is a maladaptive response with poor outcomes and limited treatment options. The transient receptor potential melastatin 4 (TRPM4) ion channel is key to activation of a Ca2+/calmodulin-dependent kinase II (CaMKII)-reliant hypertrophic signaling pathway after pressure overload, but TRPM4 is neither stretch-activated nor Ca2+-permeable. Here we show that Piezo1, which is both stretch-activated and Ca2+-permeable, is the mechanosensor that transduces increased myocardial forces into the chemical signal that initiates hypertrophic signaling via a close physical interaction with TRPM4. Cardiomyocyte-specific deletion of Piezo1 in adult mice prevented activation of CaMKII and inhibited the hypertrophic response: residual hypertrophy was associated with calcineurin activation in the absence of its usual inhibition by activated CaMKII. Piezo1 deletion prevented upregulation of the sodium–calcium exchanger and changes in other Ca2+ handling proteins after pressure overload. These findings establish Piezo1 as the cardiomyocyte mechanosensor that instigates the maladaptive hypertrophic response to pressure overload, and as a potential therapeutic target. Yu et al. show that Piezo1 is the stretch-activated mechanosensor that provides the calcium source to activate TRPM4 and the downstream CaMKII-HDAC4-MEF2 pathway, a key mediator in the cardiomyocytes’ hypertrophic response in pressure overload models.
... Dooku1 s'est par contre révélé incapable de bloquer l'activation mécanique de PIEZO1, ce qui en restreint l'utilisation expérimentale. 346 La découverte récente d'un nouvel inhibiteur de l'activation chimique de PIEZO1 par Yoda1, le Tubeimoside 1 (TBMS1) augure l'enrichissement de cette pharmacopée au cours des prochaines années. 347 ...
Thesis
La stomatocytose héréditaire à cellules déshydratées, ou xérocytose héréditaire (XH) est une pathologie autosomique dominante de la membrane des globules rouges, essentiellement causée par des mutations "gain de fonction" de PIEZO1. PIEZO1 est un canal ionique mécanosensible capable de transformer un stimulus mécanique en un influx de calcium. Nous avons découvert que PIEZO1 est précocement exprimé au cours de l'érythropoïèse. Pour la première fois, nous montrons que l'activation de PIEZO1 ralentit significativement la différenciation érythroïde, à la fois des lignées cellulaires et des cellules primaires humaines. Cet effet est spécifique de PIEZO1 puisqu'il est bloqué par un knockdown spécifique via shRNA. Dans les cellules primaires humaines, PIEZO1 maintient plus longtemps les cellules érythroïdes à un stade immature, et favorise la transcription de gènes associés à l'érythropoïèse précoce, avec majoration du rapport GATA2/GATA1 et diminution de l'expression des α/β-globines. L'activation de PIEZO1 réduit la prolifération cellulaire, avec une accumulation de cellules en phase G0/G1 du cycle cellulaire. L'effet médié par PIEZO1 requiert un influx calcique, et l'activation des voies NFAT et ERK1/2 en aval. Dans les cellules érythroïdes primaires, l'activation de PIEZO1 potentialise la réponse à l'EPO pour activer STAT5 et ERK, ce qui suggère une modulation des voies de signalisation en aval de l'EPO-R. Le retard de différenciation érythroïde in vitro a été confirmé de façon hétérogène chez 14 patients présentant une mutation PIEZO1, issus de 11 familles, porteurs de 10 mutations différentes. Nous explorons actuellement les voies dépendantes du calcium potentiellement impliquées en aval de PIEZO1, et la PKCalpha représente un bon candidat pour établir le lien entre le calcium et la signalisation EPO-dépendante. Nos données soulignent un nouveau rôle pour PIEZO1 au cours de l'érythropoïèse, qui révèle ainsi de nouvelles connaissances sur la physiopathologie des XH
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Approximately 10% of US births deliver preterm before 37 weeks of completed gestation. Premature infants are at risk for life‐long debilitating morbidities and death, and spontaneous preterm labour explains 50% of preterm births. In all cases existing treatments are ineffective, and none are FDA approved. The mechanisms that initiate preterm labour are not well understood but may result from dysfunctional regulation of quiescence mechanisms. Human pregnancy is accompanied by large increases in blood flow, and the uterus must enlarge by orders of magnitude to accommodate the growing fetus. This mechanical strain suggests that stretch‐activated channels may constitute a mechanism to explain gestational quiescence. Here we identify for the first time that Piezo1, a mechanosensitive cation channel, is present in the uterine smooth muscle and microvascular endothelium of pregnant myometrium. Piezo is downregulated during preterm labour, and stimulation of myometrial Piezo1 in an organ bath with the agonist Yoda1 relaxes the tissue in a dose‐dependent fashion. Further, stimulation of Piezo1 while inhibiting protein kinase A, AKT, or endothelial nitric oxide synthase mutes the negative inotropic effects of Piezo1 activation, intimating that actions on the myocyte and endothelial nitric oxide signalling contribute to Piezo1‐mediated contractile dynamics. Taken together, these data highlight the importance of stretch‐activated channels in pregnancy maintenance and parturition, and identify Piezo1 as a tocolytic target of interest. Spontaneous preterm labour is a serious obstetric dilemma without a known cause or effective treatments. Piezo1 is a stretch‐activated channel important to muscle contractile dynamics. Piezo1 is present in the myometrium and is dysregulated in women who experience preterm labour. Activation of Piezo1 by the agonist Yoda1 relaxes the myometrium in a dose‐dependent fashion, indicating that Piezo1 modulation may have therapeutic benefits to treat preterm labour. Abstract figure legend Proposed pathway for Piezo1‐mediated quiescence in human myometrium. Graphic created with BioRender.com.
Article
Piezo1 channels are essential mechanically activated ion channels in vertebrates. Their selective activation by the synthetic chemical activator Yoda1 opened new avenues to probe their gating mechanisms and develop novel pharmaceuticals. Yet, the nature and extent of Piezo1 functions modulated by this small molecule remain unclear. Here we close this gap by conducting a comprehensive biophysical investigation of the effects of Yoda1 on mouse Piezo1 in mammalian cells. Using calcium imaging, we first show that cysteine bridges known to inhibit mechanically evoked Piezo1 currents also inhibit activation by Yoda1, suggesting Yoda1 acts by energetically modulating mechanosensory domains. The presence of Yoda1 alters single-channel dwell times and macroscopic kinetics consistent with a dual and reciprocal energetic modulation of open and shut states. Critically, we further discovered that the electrophysiological effects of Yoda1 depend on membrane potential and temperature, two other Piezo1 modulators. This work illuminates a complex interplay between physical and chemical modulators of Piezo1 channels.
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Background Mutations in PIEZO1 cause human lymphatic malformations. We have previously uncovered an Orai1-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. Methods Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. Results Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as Klf2 , Dtx1 , Dtx3L , and Notch1 , which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. Conclusions Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.
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The Piezo channel family, including Piezo1 and Piezo2, includes essential mechanosensitive transduction molecules in mammals. Functioning in the conversion of mechanical signals to biological signals to regulate a plethora of physiological processes, Piezo channels, which have a unique homotrimeric three-blade propeller-shaped structure, utilize a cap-motion and plug-and-latch mechanism to gate their ion-conducting pathways. Piezo channels have a wide range of biological roles in various human systems, both in vitro and in vivo. Currently, there is a lack of comprehensive understanding of their antagonists and agonists, and therefore further investigation is needed. Remarkably, increasingly compelling evidence demonstrates that Piezo channel function in the urinary system is important. This review article systematically summarizes the existing evidence of the importance of Piezo channels, including protein structure, mechanogating mechanisms, and pharmacological characteristics, with a particular focus on their physiological and pathophysiological roles in the urinary system. Collectively, this review aims to provide a direction for future clinical applications in urinary system diseases.
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A short-term increase in ventricular filling leads to an immediate (Frank-Starling mechanism) and a slower (Anrep effect) rise in cardiac contractility, while long-term increased cardiac load (e.g., in arterial hypertension) decreases contractility. Whether these answers to mechanical tension are mediated by specific sensors in cardiomyocytes remains elusive. In this study, the piezo2 protein was evaluated as a potential mechanosensor. Piezo2 was found to be upregulated in various rat and mouse cardiac tissues upon mechanical or pharmacological stress. To investigate its function, C57BL/6J mice with homozygous cardiomyocyte-specific piezo2 knockout [Piezo2-KO] were created. To this end, α-MHC-Cre mice were crossed with homozygous “floxed” piezo2 mice. α-MHC-Cre mice crossed with wildtype mice served as controls [WT-Cre+]. In cardiomyocytes of Piezo2-KO mice, piezo2 mRNA was reduced by > 90% and piezo2 protein was not detectable. Piezo2-KO mice displayed no morphological abnormalities or altered cardiac function under nonstressed conditions. In a subsequent step, hearts of Piezo2-KO or WT-Cre+-mice were stressed by either three weeks of increased afterload (angiotensin II, 2.5 mg/kg/day) or one week of hypercontractility (isoprenaline, 30 mg/kg/day). As expected, angiotensin II treatment in WT-Cre+-mice resulted in higher heart and lung weight (per body weight, + 38%, + 42%), lower ejection fraction and cardiac output (− 30%, − 39%) and higher left ventricular anterior and posterior wall thickness (+ 34%, + 37%), while isoprenaline led to higher heart weight (per body weight, + 25%) and higher heart rate and cardiac output (+ 24%, + 54%). The Piezo2-KO mice reacted similarly with the exception that the angiotensin II-induced increases in wall thickness were blunted and the isoprenaline-induced increase in cardiac output was slightly less pronounced. As cardiac function was neither severely affected under basal nor under stressed conditions in Piezo2-KO mice, we conclude that piezo2 is not an indispensable mechanosensor in cardiomyocytes.
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The mechanosensitive Piezo channels function as key eukaryotic mechanotransducers. However, their structures and mechanogating mechanisms remain unknown. Here, we determine the three-bladed, propeller-like cryo-EM structures of mouse Piezo1 and functionally reveal its mechanotransduction components. Despite the lack of sequence repetition, we identify 9 repetitive units constituted of 4 transmembrane (TM) helices each, termed THUs, assembling into the highly curved blade-like structure. The last TM encloses a hydrophobic pore, followed by three intracellular fenestration sites and side portals comprising pore-property-determining residues. The central region forms a 90 Å-long intracellular beam-like structure, which undergoes a lever-like motion for connecting THUs to the pore via the interfaces of the C-terminal domain, anchor-resembling domain and outer helix. Deleting extracellular loops in the distal THUs or mutating single residues in the beam impairs the mechanical activation of Piezo1. Overall, Piezo1 possesses an unprecedented 38-TM topology and designated mechanotransduction components, enabling a lever-like mechanogating mechanism.
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Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.
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The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes.
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Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.