The biophysics of piezo1 and piezo2 mechanosensitive channels

    The biophysics of piezo1 and piezo2 mechanosensitive channels Luca Soattin, Michele Fiore, Paola Gavazzo, Federica Viti, Paolo Facci, Roberto Raiteri, Francesco Difato, Michael Pusch, Massimo Vassalli PII: DOI: Reference:

S0301-4622(15)30018-1 doi: 10.1016/j.bpc.2015.06.013 BIOCHE 5840

To appear in:

Biophysical Chemistry

Received date: Accepted date:

15 May 2015 29 June 2015

Please cite this article as: Luca Soattin, Michele Fiore, Paola Gavazzo, Federica Viti, Paolo Facci, Roberto Raiteri, Francesco Difato, Michael Pusch, Massimo Vassalli, The biophysics of piezo1 and piezo2 mechanosensitive channels, Biophysical Chemistry (2015), doi: 10.1016/j.bpc.2015.06.013

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The biophysics of piezo1 and piezo2 mechanosensitive channels

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Luca Soattina , Michele Fiorea , Paola Gavazzoa , Federica Vitia , Paolo Faccia , Roberto Raiteric , Francesco Difatob , Michael Puscha , Massimo Vassallia,∗ a Institute

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of Biophysics, National Research Council, Genova, Italy and Brain Technologies, Istituto Italiano di Tecnologia, Genova, italy c Department of Informatics, Bioengineering, Robotics, and System Engineering, University of Genova, Genova, Italy

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The ability to sense mechanical stimuli and elaborate a response to them is a fundamental process in all organisms, driving crucial mechanisms ranging from cell volume regulation up to organ development or regeneration. Nevertheless, only in few cases the underlying molecular players are known. In particular, mammals possess a large variety of mechanoreceptors, providing highly specialized functions in sensory cells, but also several housekeeping molecular systems are involved in the complex mechanism of mechanotransduction. Recently, a new class of almost ubiquitous membrane channels has been identified in mammalians, namely piezo1 and piezo2, that is thought to play a crucial role in the mechanobiology of mammals. This review focuses on recent findings on these novel channels, and highlights open biophysical questions that largely remain to be addressed. Keywords: piezo1, piezo2, mechanobiology, mechanotransduction, mechanoreceptor

1. Introduction

Biological mechanosensation is at the basis of many fundamental human functions such as touch perception, proprioception, organ development, osmotic homeostasis, hearing and equilibrioception, blood pressure regulation, bone remodeling and other physiological processes, but little is known about the molecular identity of the mechano-sensitive cellular components. In many cases, mechanical stimuli are sensed by ion channels located in the plasma membrane of sensory cells (Brierley, 2010), which could, in principle, transmit the mechanical force through a change of the bilayer tension or via the cytoskeleton (Brierley, 2010; Sukharev and Sachs, 2012). Among all ∗ Corresponding

author Email address: [email protected] (Massimo Vassalli)

Preprint submitted to Biophysical Chemistry

May 15, 2015

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mechanosensitive channels, probably the best studied ones are the microbial mechanosensitive MscS and MscL channels that appear to be activated directly by the bilayer tension (Kung et al., 2010; Booth and Blount, 2012). However, no homologues of these channels are found in metazoans (animals). Several ion channels of known function have been shown to respond to mechanical stimuli (Brierley, 2010). These include for example the classical Shaker potassium channels (Laitko et al., 2006) or some types of two-pore domain potassium (K2P) channels (Brohawn et al., 2014; Dong et al., 2015). However, the physiological role of these channels in mechanotransduction is not fully clear (Enyedi and Czirjak, 2010). Considerable progress has been made in establishing important roles of channels of the DEG/ENac and Trp families for mechanotransduction in Caenorhabditis elegans and Drosophila melanogaster (Geffeney and Goodman, 2012). However, their function as mechanotransducers in mammals is not clearly established (Geffeney and Goodman, 2012). One of the most sought-for mechanosensitive ion channels is the transduction channel of the inner ear hair cells that transduces nanometer hair cell cilia deflection in electrical hair cell activity which underlies sound perception (Effertz et al., 2015). Recently the putative TMC1 channel, which is mutated in hereditary forms of deafness, and its homologue TMC2 have been proposed as the molecular correlates of the hair cell transduction channel (Kawashima et al., 2011). However, their identity as the transduction channel is still a matter of debate (Effertz et al., 2015). A major recent advancement in the study of mechanosensation was the molecular identification of piezo1, the ion channel that underlies the currents induced by mechanical stimulation of dorsal root ganglion neurons and N2A cells, and its close homologue, piezo2, which also mediates mechanosensitive currents in heterologous expression systems (Coste et al., 2010). The identification of piezo1 and piezo2 as the first mammalian ”professional” mechanosensitive ion channels has led to a series of studies regarding their role in physiology and pathophysiology as well as their biophysical properties. The present short review summarizes the current knowledge on the molecular mechanisms underlying piezo channel activation, the methods used to study mechanosensitive ion channels and highlights a series of important questions that have to be addressed in future studies. 2. Discovering piezo1 and piezo2 channels 2.1. Identification and cloning Ion channels involved in mechanotransduction were searched by screening several cell lines and looking for mechano-activated currents similar to those previously recorded from primary DRG neurons (Coste et al., 2006). Among all, mouse neuroblastoma Neuro2A (N2A) cells proved to be an interesting mechanosensitive model. In fact, N2A cells exhibited large whole-cell currents when mechanically stimulated by a glass pipette and also large currents in cell

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Fam38a Fam38b CELE C10C5.1 Piezo

Dr

51

E7FD74 DANRE

291

2,538

26

Dr

9

E7FFV2 DANRE

282

2,444

29

51

—

340

2,955

—

40 54 35 34

E1BX07 CHICK F1NVW5 CHICK Q17897 CAEEL PIEZO DROME

322 283 276

2,485 2,793 2,438

33 34 32

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2,551

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Dr Gg Gg Ce Dm

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Hs Hs Mm Mm

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LOC100534973

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FAM38A FAM38B Fam38a Fam38b im:7149048 ENSDARG00000076870 piezo2a piezo2a.1 ENSDARG00000089035 piezo2b piezo2 ENSDARG00000076722 — — — DmPiezo

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2,521 2,752 2,547 2,822

#Exons

Table 1: Structural features of piezo proteins and genes in selected organisms. (Hs: Homo sapiens; Mm: Mus musculus; Dr: Danio rerio; Gg: Gallus gallus; Ce: Caenorhabditis elegans; Dm: Drosophila melanogaster ). Molecular weight (MW) has been calculated by ExPASy (Gasteiger et al., 2005). For entries marked with ∗ , reported information was integrated with data from the ZFIN database (Bradford et al., 2010) and the NCBI Protein database (Wheeler et al., 2007). For Danio rerio records, to provide uniqueness, the Ensemble ID was also listed. The column ’#TMD’ contains the estimated number of transmembrane domains.

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piezo2

#TMD

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piezo1

MW [kDa] 286 318 292 325

#aa

51 56 52 55

Uniprot protein name PIEZ1 HUMAN PIEZ2 HUMAN PIEZ1 MOUSE PIEZ2 MOUSE

Alias

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NCBI gene symbol PIEZO1 PIEZO2 Piezo1 Piezo2

attached patches upon the application of a negative pressure (Coste et al., 2010). Among transcripts which were highly expressed in N2A cells and encoding proteins spanning the membrane at least two times, a pool of 78 proteins was first selected. Then, using small interfering RNAs (siRNA) targeted at these transcripts and testing for the attenuation of the mechanically induced whole cell current, the Fam38A gene (Family with sequence similarity 38) was identified as a putative mechanosensitive protein (Coste et al., 2010). Indeed, once overexpressed by transfection in HEK 293, C2C12 or N2A cells, the Fam38 full length cDNA was able to produce large currents, similar to the endogenous mechanoactivated current of N2A. The Fam38A gene was thus renamed PIEZO1, from the Greek πιǫση, p´ıesi, meaning pressure. However, piezo1 is scarcely expressed in DRG cells, and it could not account for the mechano-activated current recorded from DRG (Coste et al., 2006, 2010). Indeed, Fam38B, closely related to Fam38A, was identified and cloned from DRG cDNA libraries (Coste et al., 2010). The Fam38B gene was similarly named PIEZO2, and downregulation of piezo2 in DRG neurons reduced mechanosensitive ion currents in these cells (Coste et al., 2010).

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DrPiezo2b

DmPiezo

HsPiezo1

HsPiezo2

MmPiezo1

MmPiezo2

X — — — — — — —

46 X — — — — — —

39 63 X — — — — —

26 28 34 X — — — —

57 45 38 25 X — — —

44 71 59 28 43 X — —

56 48 51 27 81 44 X —

40 67 59 35 41 88 39 X

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DrPiezo2a

DrPiezo1 DrPiezo2a DrPiezo2b DmPiezo HsPiezo1 HsPiezo2 MmPiezo1 MmPiezo2

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2.2. Phylogenetic analysis

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Table 2: Identity scores obtained from NCBI BlastP tool (Johnson et al., 2008). Identity is the number of amino acids (expressed as %) that exactly match between two different sequences, without considering gaps and where the measurement is relational to the shorter of the two sequences. Similarity over 50% is highlighted in bold; under 35% in italic. Protein access numbers are as follows: DrPiezo1: XP 696355.4; DrPiezo2a: 002666625; DrPiezo2b: 003198010; DmPiezo1: AFB77909.1; HsPiezo1: NP 001136336.2; HsPiezo2: NP 071351.2; MmPiezo1: NP 001032375.1; MmPiezo2: NP 001034574.4.

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Primordial mechanosensitive structures probably emerged as protective adaptations to osmotic stress (Levina, 1999; Booth and Blount, 2012) and may have been coopted in a wide set of specialized tasks such as cellular homeostasis, hearing, discriminative touch or nociceptive sensitivity (True and Carroll, 2002; Chalfie, 2009). Piezo genes and proteins have been identified in various organisms, surviving a strong evolutionary pressure that contributed to maintain sequences homology between different organisms. Piezo is considered a mechanotransduction model, well-preserved among phylogenetically distant species (Coste et al., 2010). A list of features collected from NCBI Gene (Brown et al., 2014) and UniProt databases (Consortium, 2014) is presented in table 1 for the most studied organisms. Commonly, animals, plants and eukaryotes contain a single piezo, whereas vertebrate organisms such as chicken (Gallus gallus), mouse (Mus musculus) and humans (Homo sapiens) have two members, called piezo1 and piezo2. As shown in Coste et al. (2010), piezo gene duplication seems to appear in the passage between early chordates (Ciona intestinalis) and vertebrates. Interestingly, in zebrafish (Danio rerio) the presence of 3 piezo encoding genes is reported, in accordance with a general phenomenon associated to genome duplication in teleost fishes during their evolutionary history (Hultman et al., 2007). Moreover, multiple piezo proteins have been identified in the Ciliophora kingdom: the protozoan Tetrahymena thermophila has three members and Paramecium tetraurelia no less than six, while no homologous were identified in yeast or bacteria (Coste et al., 2010; Prole and Taylor, 2013). Homology data are shown in table 2, where percentage of protein sequence identity among the most studied species is reported.

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Human Piezo1

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Human Piezo2

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Figure 1: Putative topology of PIEZ1 HUMAN (top) and PIEZ2 HUMAN (bottom). FASTA sequences were obtained from UniProt and processed by Protter webclient, ETH-Zurich (Omasits et al., 2013). Although similar trends were evident, different algorithms implemented in diverse webtools predicted different numbers of transmembrane helices, between 25 and 40, with differences in the predicted orientation of some extramembrane loops. The red box highlights the CTL2 loop, involved in the gating kinetics of the channel (see section 3.1) and homologous to the crystallized fragment from C.elegans (see figure 2).

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2.3. Structure Piezo proteins show no recognizable sequence similarity to other known proteins, and internal sequence repeats within piezo subunits have not been detected. Piezo proteins are composed by about 2500-2800 amino acids and are predicted to encompass between 26 and 40 transmembrane domains (see table 1) (Kamajaya et al., 2014). Noticeably, human piezo1 and piezo2 are the two human proteins predicted to show the highest number of transmembrane segments (Volkers et al., 2014). Putative topologies of human piezo1 and human piezo 2 are depicted in figure 1, as obtained by predictive modeling (Omasits et al., 2013). Due to the large size of piezo proteins, crystallographic analyses represent a formidable technical challenge. Recently, Kamajaya and coworkers solved the three-dimensional structure of the soluble C-terminal loop 2 (CTL2) from the Caenorhabditis elegans piezo. The loop is located just before the last putative transmembrane helix and the C-terminal tail (see figure 1) (Kamajaya et al., 2014). CTL2 is the largest conserved loop (about 250 aa) among piezo homologs, showing a moderate degree of sequence conservation that suggests a key role in the protein functionality. Moreover, point mutations in the CTL2 domain of human piezo proteins are involved in the gating kinetics of the channel (Bae et al., 2011). The CTL2 model published by Kamajaya et al. (2014) establishes that the region adopts a topologically distinct β-sandwich fold with three sheets, where sheet 1 is nearly parallel to sheet 2 and perpendicular to sheet 3 (see figure 2). This fold does not appear to be shared with any other family of proteins in eukaryotes, thus contributing to highlight the distinctive nature of 5

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piezo family (Kamajaya et al., 2014). It is worth noting that in human piezo1 CTL2, the substitution of methionine with arginine in position 2225 (M2225R) causes dehydrated hereditary stomatocytosis (DHS) disease (see table 3). The structure of the C. elegans CTL2 containing the equivalent mutation (M31R) resulted very similar to the wild type, besides a shift of about 3˚ A of the arginine R94 side chain away from arginine R31 (Kamajaya et al., 2014). This suggests that the mutation does not affect folding, but may perturb more slightly energetics, through changes in electrostatics, or may interfere with the protein ability to interact with other interface regions.

Figure 2: Crystal structure of the wild type CePiezo CTL2 β-sandwich fold (PDB File 4PKX). The β-sheets are numbered sequentially (1,2 and 3) from the N-terminus. Picture created with UCSF Chimera.

Several hypotheses are being investigated to evaluate the functional assembly of the protein in the plasma membrane. Total Internal Reflection Fluorescence (TIRF) studies have been performed on eGFP-fused MmPiezo1 expressed in Xenopous laevis. The analysis of single spot fluorescence indicated the predominant presence of four discrete photobleaching steps, suggesting a homotetrameric architecture (Coste et al., 2012). The same conclusion was obtained using biochemical approaches, showing the appearance of a band corresponding to a tetramer with a total molecular weight of about 1236 kDa (Coste et al., 2012). The supramolecular organization of the functional piezo unit is expected to be tightly linked with its ability to respond to external mechanical stimuli, but the details of this mechanism have not yet been fully disclosed. In general, the activation and inactivation of the currents seem to be associated with the presence of whole tetramers, but two different perspectives have been proposed 6

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(Gottlieb et al., 2012; Bae et al., 2013a). One possible picture is that one central pore is formed at the boundary of the monomers. This model simply explains some aspects, such as the need for tetramers and the sensitivity of the complex to membrane stretch (see section 3). Alternatively, each monomer in the tetrameric assembly can contain its proper ion conducting pore, similarly to aquaporins. In this case, mechanosensitivity could be an independent function of each monomer or could be related to the tetrameric assembly (Bae et al., 2013a).

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2.4. Disease-associated genetic variants Human PIEZO1 and PIEZO2 are highly polymorphic genes: for both of them, the 1.000 Genomes database (Durbin et al., 2010) reports evidence of thousands of variants, including 495 missense variants, 11 stop gained mutations and more than 30 in-frame insertions or deletions for PIEZO1, and 1639 missense and 62 stop gained variants for PIEZO2. Some of the variants are predicted as deleterious, and have been found associated with human pathologies. In particular, hereditary xerocytosis, also known as dehydrated hereditary stomatocytosis (DHS, OMIM 194380), an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration, is caused by PIEZO1 mutations, as reported in table 3. It has been speculated that the erythrocyte dehydration is due to an imbalance in intracellular cation concentrations. This might suggest that piezo1 plays a role in erythrocyte volume regulation. On the other hand, PIEZO2 mutations have been found associated with distal arthrogryposis (DA) forms, in particular to type 5 (OMIM 108145, type 3 (OMIM 114300) and Marden-Walker syndrome (OMIM 248700), probably representing variable expressivity of the same disease (McMillin et al., 2014), characterized by generalized autosomal dominant contractures with limited eye movements, restrictive lung disease, and variable absence of cruciate knee ligaments. 3. Electrophysiological properties 3.1. Gating Coste et al. (2010) activated piezo1/2 mediated currents by applying mechanical force on the cell surface using a glass pipette probe (indentation) in the whole-cell patch clamp configuration (Figure 3A), and also by applying suction (negative pressure) to the membrane in the cell-attached configuration (Fig. 3B). A negative pressure of −60mmHg yielded maximal channel opening. In both cases, mechanical stimuli reversibly induced large currents. In all cells overexpressing piezo1, the current-voltage relationship was linear between −80mV and +80mV . In the cell-attached configuration the single-channel conductance evoked by negative pressure was about 23pS. Importantly, current mediated by both piezo channels was transient, i.e. it inactivated almost completely in the continued presence of the mechanical stimulus. Moreover, the kinetics of 7

DHS

E2496ELE

DHS

I802F

–

X

–

X

slower inactivation slower inactivation slower inactivation slower inactivation

slower inactivation increased cation transport in erythroid cells slower inactivation recurrent mutation

Human piezo2 X X

DA5

–

–

DA5 DA5 DA5 DA5 DA3, DA5 MWKS DA5 DA5

X X X – X X – X

– X – X X – X –

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M998T T2221I S2223L T2356M R2686H R2686C R2718L R2718P

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M712V

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DHS DHS DHS DHS DHS DHS DHS DHS DHS DHS DHS DHS

Inherited

Human piezo1 – X – X – X – X – – – X – X – X – X – X – – – X

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G718S G782S R808Q S1117L R1358P A2003D A2020T A2020V T2127M K2166-2169del M2225R R2456H

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E2727del

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X

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Y2737Ifs*7 S2739P W2746*

DA5 DA5 DA5

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1 1 1 1 2 1 2 1 1,2 1 2-4 1-6 1 2 7

faster recovery from inactivation

recurrent mutation

recurrent mutation. In-frame deletion. Slower inactivation Faster recovery from inactivation

Table 3: Mutations in human piezo1 and piezo2, associated with human pathologies. PIEZO1 and PIEZO2 genes are located in chromosome 16 and 18, respectively. References are: 1 (Andolfo et al., 2013); 2 (Albuisson et al., 2013); 3 (Zarychanski et al., 2012); 4 (Bae et al., 2013a); 5 (Shmukler et al., 2014); 6 (Beneteau et al., 2013); 7 (McMillin et al., 2014); 8 (Coste et al., 2013)

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8 7 7 7 7 7 7 7 7 7,8 7 7 7

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Figure 3: Current traces measured in HEK293T cells overexpressing piezo1 channels evoked by indentation (A, whole cell configuration) or by negative pressure (B, cell-attached configuration). Figure adapted from (Coste et al., 2010).

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inactivation of piezo2 mediated currents was faster than that of piezo1 mediated currents. Inactivation time constant of piezo1 expressed in HEK293 cells was about 15ms at −80mV and about 7ms for piezo2 whole cell currents (Coste et al., 2010). From a more detailed kinetic analysis, Gottlieb et al. (2012) proposed that piezo1 gating kinetics could be well fit by a 3 states model: closed, open and inactivated. However, repeated cell stimulations by high pressure (positive or negative) and high voltage led to a gradual decrease of the degree of inactivation (Gottlieb et al., 2012).

3.2. Ion selectivity Both piezo1 and piezo2 channels are cation selective with a selectivity sequence of Ca2+ > K+ > Na+ > Mg2+ (Coste et al., 2010). In cell-attached patches, divalent ions reduced the single channel conductance by about 50% for Mg2+ , Ca2+ and Zn2+ (Coste et al., 2012; Gottlieb et al., 2012). In addition, divalent cations slowed the time course of inactivation (Gottlieb et al., 2012). Moreover, in whole-cell recording, extracellular divalent ions were reported to be required for channel activity evoked by cell indentation (Gottlieb et al., 2012). 3.3. Inhibitors Several compounds, like ruthenium red and gadolinium, are known to inhibit mechanosensitive cation channels in various systems (Coste et al., 2010). Interestingly, 30µM ruthenium red or 30µM gadolinium were able to block about 80% of both piezo1 and piezo2 mediated currents (Coste et al., 2010; Drew et al., 2002; Hao et al., 2009). Furthermore, it was shown that also the GsMTx4 (Grammostola spatulata mechanotoxin 4) peptide is able to reversibly inhibit more than 80% of piezo1 mediated currents in outside-out patches from transfected HEK293 cells. GsMTx4 was isolated from a tarantula venom and is the only known peptide inhibitor of cationic mechanosensitive ion channels (Gottlieb et al., 2007; Bowman et al., 2007; Bae et al., 2011). GsMTx4 specifically 9

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Figure 4: Inhibitor compounds. Reversible effect of Ruthenium red and gadolinium on mechanically activated piezo1 currents in HEK293 cells (A, B) and of GsTMx4 (C). Figure adapted from (Coste et al., 2010; Bae et al., 2011).

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inhibits piezo1 from the extracellular side, as internal application of GsMTx4 to inside-out patches did not inhibit channel activity (Bae et al., 2011). GsMTx4 has been proposed to be a gating modifier for piezo1 (see figure 4).

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Some of the disease causing mutations have been functionally analyzed in heterologous expression systems (see table 3). A common feature of many of these mutations is a significant slowing of the time course of inactivation and an introduction of a latency of current activation after the mechanical stimulus (Bae et al., 2013a; Albuisson et al., 2013). However, the peak currents recorded from mutants and wild type were similar, with the exception of currents induced by M2225R, which were overall larger (see figure 6A). Interestingly, combining M2225R with R2456K mutation resulted in an almost complete lack of inactivation, while other channel properties remained similar to wild type (Bae et al., 2013b). Single-channel recording of M2225R, R2456H and R2456K mutants (the latter being not associated to diseases) showed a pronounced latency to activation (Bae et al., 2013a). Measurements of the pressure response curve in the cell-attached configuration for piezo1 wild type and the same mutants (M2225R, R2456H, and R2456K) revealed a left shift of the half-maximal pressure of activation, P1/2 , for R2456H and R2456K, while M2225R behaves similarly to wild type (Bae et al., 2013a). Since a slower inactivation leads to an overall increased cation influx, the piezo1 xerocytosis causing mutations can be considered a gain of function (Albuisson et al., 2013), consistent with the dominant inheritance. The mutations do not seem to act via change in residue charge because, for instance, the conservative (not disease related) mutation of arginine (R) to lysine (K) at position 2456 slows down inactivation more than the charge changing mutation R2456H (Bae et al., 2013a). In a parallel study (Andolfo et al., 2013), the R2488Q and R2456H mu-

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Figure 5: Several biophysically characterized mutants associated with disease exhibit a slowed time course of inactivation. Example currents traces from overexpressing HEK293 cells are shown for piezo1 (A) and piezo2 (B) mutants. Figure adapted from (Albuisson et al., 2013; Coste et al., 2013)

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tants were expressed in Xenopus laevis oocytes. In on-cell patches, hydrostatic pressure-induced currents were increased for R2488Q mutant but not for R2456H. Interestingly, the single channel conductance of mutant R2456H was almost doubled compared to WT (43pS vs. 25pS). However, no controls for channel identity were performed in that study and it has to be kept in mind that Xenopus oocytes are known to exhibit relatively large endogenous stretchactivated channel activity (Methfessel et al., 1986). Other on-cell patch experiments on red blood cells from xerocytosis affected patients showed spontaneous ion-channel activity with a single-channel conductance of about 13pS. No spontaneous channel activity was seen in cells from control individuals. The channel activity was blocked by 2.5µM of GsTMx4 in the pipette (Andolfo et al., 2013). It is interesting to observe that several xerocytosis causing mutations are located in the C terminal half of the protein (see table 3). Removing 303 C terminal amino acids by introducing a stop codon at position G2218, resulted in channels that exhibits a long latency, slowing or complete loss of inactivation, and very slow deactivation (Bae et al., 2013a). Probably, the C terminal region is implicated in piezo1 gating kinetics (Albuisson et al., 2013). Moreover, the C terminus could be involved in the tetrameric assembly or in the interaction with not yet identified ligands or other proteins (Kamajaya et al., 2014). Two piezo2 mutants (I802F and E2727del) have been characterized in heterologous systems (Coste et al., 2013). They exhibited mechanically activated currents of similar amplitude compared with wild type currents and a linear current-voltage relationship. Only for the truncated mutant (E2727del) a slowing of the inactivation kinetics was described, which, as for the piezo1 mutants,

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would result in a larger cation influx. Interestingly, assaying the ability to respond to repetitive stimulation, both mutants showed a ∼ 2-fold faster recovery from inactivation (see figure 6).

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4. Models of mechanical activation

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The ability to sense mechanical stimuli and to convert them into a biological response (mechanotransduction) is a key recognized mechanism in sensory cells (Lumpkin and Caterina, 2007), but it recently emerged as an ubiquitous process, with a profound relevance in development and homeostasis in all organisms (Wong et al., 2011). Mechanically gated ion channels are among the main players of mechanotransduction, providing the ability to directly convert mechanical signals into ionic fluxes. To have a deeper insight into mechanotransduction, detailed information on mechanical channel gating is required.

Figure 6: Two different mechanical gating mechanisms are being evaluated: [top] pure bilayer tension transmission; [bottom] tether model. Figure adapted from Haswell et al. (2011)

Mechanoreceptors are efficient sensors, tightly coupled to their environment, able to sense forces and stresses in their near proximity. Two main models have been proposed to account for this ability (see Figure 6), together with the possibility to have indirect channel gating mediated by a non-channel mechanosensor exploiting one of these two mechanisms (Lumpkin and Caterina, 2007). The molecular origin of force sensing in piezo1 and piezo2 channels is still elusive, but some information has already been gathered. One of the main questions regarding channel gating (activation / inactivation) relates to the involvement of the cytoskeleton. The role of actin was explored by Gottlieb et al. (2012), who measured mechanically evoked currents in the cell-attached configuration on HEK293 cells overexpressing piezo1, before and after the treatment with cytochalasinD (cytoD), which disrupts f-actin. CytoD only reduced the steady-state response during inactivation, leading to a more complete inactivation. However, in the whole cell configuration cytoD produced a pronounced reduction in current amplitude suggesting that the stress in actin is important for the activation of the channel.

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A relevant result was recently obtained regarding the role of an associated protein of the stomatin family, STOML3, on the mechanical gating of piezo channels (Poole et al., 2014). The stomatin domain identifies a family of ubiquitary proteins that are present in all classes of life. The function of stomatin proteins is still unclear, but it is relatively established that they oligomerize and localize to membrane domains (Lapatsina et al., 2012). The use of knockout mice for STOML3 allowed to investigate the role of this stomatin-domain protein on the onset of mechanically evoked currents in DRG neurons, showing that STOML3 is able to enhance the activation propensity of one order of magnitude (Poole et al., 2014). This finding suggests that mechanosensitivity of piezo channels is intrinsic, but the presence of local protein structures in the plasma membrane is needed to amplify the mechanism, reaching an effective level. Moreover, not only STOML3 seems to be involved in the process of clustered mechanosensitivity, but also adhesion structures are expected to play a relevant role. In fact, a smart experiment was performed by plating DRG neurons on substrates holding active nano-pillars, being able to apply controlled nanometric deformations on the cells from the substrate side. Comparing the effect of this stimulus with standard indentation experiments, it was reported that stimulation by pillar movements has a 10 times higher efficiency, compared to cell body indentation, being also able to evoke sustained currents in several neurons from stoml3−/− mice (Poole et al., 2014).

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Besides the influence of membrane protein clusters (adhesion structures and stomatin-domain proteins) on the mechanotransduction ability of piezo channels, also the physico-chemical state of the plasma membrane has a key role. In fact, one of the main inhibitors of piezo1 and piezo2 channels is the Grammostola spatulata mechano-toxin GsTMx4 (Andolfo et al., 2013). The inhibition mechanism of this peptide is not directed against the channel, but it targets the cellular membrane, putatively penetrating the lipid bilayer and creating a transbilayer coupling (Nishizawa and Nishizawa, 2007). The abrupt change in membrane mobility induced by the presence of the link would probably affect the ability of the bilayer to transfer mechanical tension to membrane proteins and domains. The data collected up to date on the mechanism of mechanotransduction of piezo channels are not enough to account for the complexity of the observed phenomena and not even a tentative model can be cast yet. As a matter of fact, it seems that several components are affecting this complex mechanism, but dissecting single factors is a challenging task, mainly due to the structural characteristics of these channels (see section 2.3) 5. Conclusions and perspectives Albeit research activity on piezo proteins has already clarified several critical aspects concerning the nature and properties of these molecules, much work is still needed to fully understand their response to mechanical stimuli and their

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mechanistic interactions within the naturally expressing organisms. The expression of piezo1 and piezo2 in a variety of mammalian cell lines induces mechanically activated cationic currents. Mechanically activated currents are elicited both as a function of positive and negative pressure stimuli on the cell membrane. Remarkably, the huge ∼ 1.2 MDa tetrameric piezo1 complex has been overexpressed in mammalian cells, purified, and reconstituted in both liposomes and asymmetric planar bilayers retaining its overall conduction properties, although altered with respect to its performances in cells (Coste et al., 2012). Such a reduced system could be extremely useful to study structural aspects of piezo complexes. In general, future investigation will have to address so far undisclosed issues such as those related to the real nature of the piezo subunits, whether mechanosensitive per se or taking advantage of still unknown interacting partners to sense membrane tension; the role of protein clusters and that of dynamic cytoskeletal interacting partners that can introduce regulatory constraints on channel activity at cell membrane; the role of the mechanical state of the membrane (e.g. local vs general) related to environmental conditions and membrane lipid composition. Closely related to this last point, a crucial aspect is the full understanding of the mechanisms of action of membrane targeting GsMTx4 in inhibiting piezo1-induced currents. From a biophysical stand-point, the most promising approach appears to be that of coupling different techniques to cope with the challenges posed by the elusive nature of these large biomolecules. Remarkable advances are likely to come from improving the mechanical stimulation set-ups towards more quantitative ways of applying pressure stimuli to cells. Last generation nano-bioindenters or NEMSbased sensors/actuators are foreseen to be crucial in that respect. They could be advantageously coupled with other techniques such as patch/voltage clamp, or fluorescence calcium imaging to measure mechanically induced calcium influxes. Also optical manipulation of bio-functionalized beads by laser tweezers holds relevant promises for quantitative, space-resolved, bio-targeted force actuation on cell bodies while observing the resulting effects by fluorescence microscopy. Systematic, robust reconstitution in lipid bilayers with varying composition could be very helpful to investigate membrane distribution and 2d arrangement of these complex molecules, the role of specific lipids and of the nanomechanical properties of the lipid bilayer in modulating the conformational/functional response of piezo channels. Such samples could enable cryo-EM/AFM high resolution imaging, fostering the retrieval of information on molecular conformation and structure, thus allowing the plausible design of a molecular model for the protein oligomer. References Albuisson, J., Murthy, S.E., Bandell, M., Coste, B., Louis-dit Picard, H., Mathur, J., F´en´eant-Thibault, M., Tertian, G., de Jaureguiberry, J.P., Syfuss, P.Y., et al., 2013. Dehydrated hereditary stomatocytosis linked to gainof-function mutations in mechanically activated piezo1 ion channels. Na14

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