ASIC toxins Baron review Toxicon mai2013

Review

Venom toxins in the exploration of molecular, physiologicaland pathophysiological functions of acid-sensing ion channels

Anne Baron a,b,c, Sylvie Diochot a,b,c, Miguel Salinas a,b,c, Emmanuel Deval a,b,c,Jacques Noël a,b,c, Eric Lingueglia a,b,c,*

aCNRS, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 7275, 06560 Valbonne, FrancebUniversité de Nice-Sophia Antipolis, 06560 Valbonne, Francec LabEx Ion Channel Science and Therapeutics, 06560 Valbonne, France

a r t i c l e i n f o

Article history:

Received 19 February 2013

Accepted 10 April 2013

Available online xxxx

Keywords:

ASIC

Sodium channels

Toxins

Nervous system

Pathophysiology

Pain

a b s t r a c t

Acid-sensing ion channels (ASICs) are voltage-independent proton-gated cation channels

that are largely expressed in the nervous system as well as in some non-neuronal tissues.

In rodents, six different isoforms (ASIC1a, 1b, 2a, 2b, 3 and 4) can associate into homo- or

hetero-trimers to form a functional channel. Specific polypeptide toxins targeting ASIC

channels have been isolated from the venoms of spider (PcTx1), sea anemone (APETx2)

and snakes (MitTx and mambalgins). They exhibit different and sometimes partially

overlapping pharmacological profiles and are usually blockers of ASIC channels, except for

MitTx, which is a potent activator. This review focuses on the use of these toxins to explore

the structure–function relationships, the physiological and the pathophysiological roles of

ASIC channels, illustrating at the same time the therapeutic potential of some of these

natural compounds.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Under the double evolutionary pressure to select thebest hunting and self-defense strategies (against predatorsor competitors), venomous animals have engineeredinnovative and unique protein structural motifs that cantarget a variety of receptors and ion channels with highaffinity and specificity. Sincemore than 40 years, venoms ofspecies like spiders, scorpions, sea anemones, snakes andcone snails have provided a rich pharmacopoeia. Withtherapeutic motivations, scientists first identified the toxiccomponents, called toxins, responsible for severe animalenvenomations (Chang, 1979; Karlsson, 1979; Mirandaet al., 1970; Mylecharane et al., 1989; Olivera et al., 1985;Renaud et al., 1986). They further identified the molecular

targets of venom toxins to understand their mode of action,and showed that some venom peptides were also of highinterest for basic science (Han et al., 2008; Harvey, 2001;McCleary and Kini, 2013; Menez, 1998; Servent et al., 2000;Twede et al., 2009). A number of venompeptides have beensuccessfully exploited for medical purposes too. Angio-tensin converting enzyme (ACE) inhibitor from the venomof the Brazilian arrowhead viper Bothrops jararaca led forinstance to a potent anti-hypertensive drug (Captopril),which is one of the most prescribed drugs in the world(Ferreira et al., 1970; Koh and Kini, 2012).

Animal toxins were key pharmacological tools in the ionchannel field to study structure–function, gating mecha-nisms and tissue localization of many channels (Dutertreand Lewis, 2010). Some of these peptides even lead toclinical development and venom-based drugs, like inrecent years ziconotide (SNX-111, Prialt), an inhibitor ofneuronal voltage-gated Ca2þ channels isolated from Conus

magus, for patients with intractable pain who do not

* Corresponding author. CNRS, Institut de Pharmacologie Moléculaire

et Cellulaire, UMR 7275, 06560 Valbonne, France.

E-mail address: [email protected] (E. Lingueglia).

Contents lists available at SciVerse ScienceDirect

Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

0041-0101/$ – see front matter � 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.toxicon.2013.04.008

Toxicon xxx (2013) 1–18

Please cite this article in press as: Baron, A., et al., Venom toxins in the exploration of molecular, physiological and patho-physiological functions of acid-sensing ion channels, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.04.008

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respond to other drugs (Miljanich, 2004; Schmidtko et al.,2010).

Acid-sensing ion channels (ASICs) were first identi-fied in the laboratory of M. Lazdunski about 15 years ago(Waldmann et al., 1997b). Six protein isoforms are codedby four genes in rodents: ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3 and ASIC4 (Akopian et al., 2000; Bassleret al., 2001; Chen et al., 1998; Garcia-Anoveros et al.,1997; Gründer et al., 2000; Lingueglia et al., 1997;Price et al., 1996; Waldmann et al., 1997a, 1997b, 1996),that associate in homo- or hetero-trimers (Jasti et al.,2007). They are activated by extracellular acidic pH tomediate a sodium-selective, amiloride-sensitive current.Interestingly, the human ASIC3 channels have beenrecently shown to be sensitive to both acidic and alka-line pH in certain conditions (Delaunay et al., 2012). TheASIC2b and ASIC4 subunits do not form functionalhomomeric proton-gated channels by themselves, butASIC2b can associate with other ASIC subunits to confernew properties on the heteromeric channels (Devalet al., 2004; Lingueglia et al., 1997). The role of ASIC4is still not clear. ASICs are largely expressed in the cen-tral and peripheral nervous systems, primarily found inneurons. ASIC1a and ASIC2 (both variants a and b) arepresent throughout the central nervous system whileASIC1b and ASIC3 are principally found, at least in ro-dents, in sensory neurons of the trigeminal, vagal, anddorsal root ganglia that also expressed ASIC1a andASIC2. Peripheral tissue acidosis associated with painoccurs in situations like inflammation, ischaemia, frac-tures, haematomas, tumours, wounds or after surgicalprocedures. ASIC channels are of particular interest sincethey are highly sensitive to moderate acidifications. Theyare for instance 10-fold more sensitive than TRPV1,another ion channel activated by protons, capsaicin andheat in nociceptive neurons. ASIC channels can generatesustained depolarizing currents upon prolongedtissue acidification compatible with the detection ofnon-adapting pain. In central nervous system, ASICchannels have been involved in several pathologicalconditions like ischaemia, seizures or neuroinflamma-tion where extracellular acidification occurs. A role insynaptic physiology has also been suggested, whichcould be relevant of the acidification of the synaptic cleftupon neuronal activity after the release of the acidiccontent of synaptic vesicles (Miesenbock et al., 1998;Ozkan and Ueda, 1998). ASIC currents and/or transcriptshave also been observed in glia, smooth muscle cells,lung epithelial cells, immune cells, urothelial cells, adi-pose cells, joint cells and osteoclasts, indicating thatASICs likely play a role in non-neuronal cells as well (forreviews on ASICs see Deval et al., 2010; Gründer andChen, 2010; Li and Xu, 2011; Noël et al., 2010; Slukaet al., 2009; Wu et al., 2012).

2. Venom toxins targeting ASIC channels

2.1. Four toxins with different structures

To date, four compounds targeting ASIC channels werepurified fromvenoms of various origins. The spider peptide

PcTx1, the sea anemone peptide APETx2, the snake pep-tides mambalgins and the snake heterodimeric proteinMitTx.

PcTx1 was the first high-affinity inhibitor (IC50w1 nM)described for ASIC channels. This 40 amino-acid peptide(Fig. 1A) has been isolated as a minor component (less than1% of protein content) of the South American tarantulaPsalmopoeus cambridgei venom (Escoubas et al., 2000). Itsstructure was first solved by NMR in 2003 (PDB code1LMM; Escoubas et al., 2003) and then further refined in2011 (PDB code 2KNI; Saez et al., 2011). It consists of athree-stranded anti parallel b-sheet, defining three loopsand a compact disulfide bonded core (Fig. 1B). PcTx1 isfolded according to the Inhibitor Cystine Knot (ICK) motif,also found in other spider and cone snail toxins acting onvoltage-gated ionic channels (Craik et al., 2001; Pallaghyet al., 1994). This cystine knot confers a resistance toextreme pH, organic solvents, and high temperatures. Thisstructure is also resistant to proteases, which could be anadvantage in regard to galenic and administration routesfor therapeutics. Despite this global structural homologywith other ICK peptides, PcTx1 has no more than 28%sequence identity with known spider toxins (Escoubaset al., 2000).

The sea anemone peptide APETx2 was the secondASIC-targeting peptide discovered in 2004, as a minor (2%of protein content) constituent in the venom of the seaanemone Anthopleura elegantissima (Diochot et al., 2004).This peptide of 42 amino acids (Fig. 1A) displays 64%sequence identity with APETx1, a blocker of HERG Kþ

channel, 34% sequence identity with BDS peptides, whichblock the KV3 voltage-gated potassium channels, and25–29% sequence identity with AP-A, AP-B, AP-C toxinsknown to activate voltage-gated sodium channels (Bruhnet al., 2001; Castaneda and Harvey, 2009; Diochot et al.,2003, 1998; Norton et al., 1978; Yeung et al., 2005). TheAPETx2 structure was determined by two-dimensional 1HNMR using the native toxin (Chagot et al., 2005) (Fig. 1B).It consists of a compact disulfide-bonded core composedof a four stranded b-sheet connected by three disulfidebonds, from which a loop (15–27) and the N- and C-termini emerge. It belongs to the disulfide-rich all-bstructural family with a fold typical of the defensin familyof peptides (Torres and Kuchel, 2004), which alsoincludes APETx1, BDS peptides, AP and ATX toxins (Navactivators).

MitTx was identified in 2011 from the venom of theTexas coral snake Micrurus tener tener (Bohlen et al., 2011).MitTx has a b-bungarotoxin-like structure with two sub-units, a MitTx-a subunit consisting of a 60 amino-acidKunitz type peptide, and a MitTx-b subunit, which is a120 amino-acid phospholipase A2-like protein (Fig. 1A, B).Both subunits are involved in a 1:1 interaction (Kdw12 nM), but this interaction is non-covalent, unlike the b-bungarotoxins that are linked by an interchain disulfidebond.

In 2012, two 57 amino-acid ASIC-targeting peptideshave been identified from the African black mamba Den-

droaspis polylepis polylepis venom (representing less than0.5% of protein content), which only differ by one residue(at position 4) and have been called mambalgin-1 and

A. Baron et al. / Toxicon xxx (2013) 1–182


mambalgin-2 (Diochot et al., 2012) (Fig. 1A). An ortho-logue peptide that differs by one amino-acid at position 23has also been recently identified in the green mambaDendroaspis angusticeps venom and has been calledmambalgin-3 (H. Schweitz and S. Diochot, personalcommunication) (Fig. 1A, Uniprot, access number#C0HJB0). Mambalgins belong to the family of three-finger toxins (3FTxs) but define a new sub-family withno more than 50% sequence identity with other snake3FTxs (Fig. 2). A model of their 3D-structure show thetypical three-finger fold, with three loops emerging from acore stabilized by four disulfide bonds, but mambalginshave shorter loop I and loop III than other 3FTxs, with onlyone finger (loop II) protruding from the core (Fig. 1B). Theyare related to the “non-conventional toxins” also referredas “weak toxins” such as CM-2a, CM-3 and CM-1b, char-acterized by a low toxicity (LD50 varying from 5 to 80 mg/kg) (Joubert and Taljaard, 1980; Utkin and Osipov, 2007)compared to the potent a-neurotoxins (LD50 from 0.04 to0.3 mg/kg) (Endo and Tamiya, 1991).

2.2. Pharmacological properties of ASIC-targeting toxins

The four ASIC-targeting toxins show different, althoughsometimes overlapping, pharmacological profiles on ASICchannels (Table 1).

Nanomolar concentrations of PcTx1 applied at physio-logical pH 7.4 potently inhibit rodent homomeric ASIC1achannels (IC50 w1 nM) (Escoubas et al., 2000). Recently,PcTx1 has also been shown to inhibit heteromeric ratASIC1a þ ASIC2b channels with similar affinity to ratASIC1a channels (IC50w3 nM) (Sherwood et al., 2011). Thehuman ASIC1a channel (i.e., the human orthologue of ratASIC1a) is inhibited by the PcTx1-containing venomapplied at pH 7.2 (Sherwood and Askwith, 2008), and byPcTx1 at pH 6.2 (IC50w13 nM) (Qadri et al., 2009). Higherconcentration of PcTx1 (60 nM) applied at a holding pH of7.4 can produce enhancement of the hASIC1a currentactivated by pH 6.7 (Hoagland et al., 2010). At high con-centration, PcTx1 also interacts with human and rodentASIC1b channel by potentiating its activation by acidifica-tion (EC50 w100 nM) (Chen et al., 2006; Hoagland et al.,2010). Finally, PcTx1 directly activates chicken ASIC1channels (EC50w189 nM) (Samways et al., 2009; Baconguisand Gouaux, 2012). The peptide has no effect on the otherhomomeric or heteromeric ASIC channels tested, as well ason a variety of Kv, Nav and Cav channels (Escoubas et al.,2000). The specificity of the toxin for ASIC channels issupported by binding experiments with an iodinated formof the peptide, which show similar binding properties onrat brain membranes and heterologously expressed chan-nels (Salinas et al., 2006).

Fig. 1. Structure of ASIC-targeting toxins. A, Sequence of the ASIC-targeting toxins that block (PcTx1, APETx2, mambalgins) or activate (MitTx) ASIC channels.

MitTx is constituted by the non-covalent association of MitTxa and MitTxb. A sequence alignment is shown for mambalgins (Mamb-1, Mamb-2 and Mamb-3).

Residues in Mamb-2 and -3 that are different from Mamb-1 are indicated by black boxes. Disulfide bonds are indicated by red lines. B, Three dimensional

structure of PcTx1 (PDB code 2KNI; Saez et al., 2011), APETx2 (PDB code 1WXN; Chagot et al., 2005) and structural models of mambalgin-1 (Diochot et al., 2012)

and MitTx (Bohlen et al., 2011). All toxins are shown at the same scale (disulphide bridges in orange). (For interpretation of the references to colour in this figure

legend, the reader is referred to the web version of this article.)

A. Baron et al. / Toxicon xxx (2013) 1–18 3


Mambalgins inhibit homomeric rodent and humanASIC1a channels, homomeric rodent ASIC1b channels aswell as other heteromeric ASIC1a-containing and ASIC1b-containing channels with IC50 ranging from 11 to 252 nM(Table 1). Mambalgin-1, -2 and -3 display the same

pharmacological profile (Diochot et al., 2012 and S. Diochotand M. Dauvois, personal communication). Mambalginsinhibit all the ASIC channel combinations expressed incentral neurons (i.e., ASIC1a, ASIC1a þ ASIC2a andASIC1a þ ASIC2b). They were accordingly found to

Fig. 2. Mambalgins define a new structural and functional class among snake three-finger toxins. Phylogenetic tree built from blast results using the mambalgin-

1 protein sequence. The most similar toxins (CM-2a, CM-3, CM-1b, OH-26) only share 45–57% sequence identity. For clarity, only the most representative toxins

are shown for each subfamily, and some branches (associated with black dots) are not expanded. Colour boxes represent toxins sharing a common functional and/

or molecular mode of action indicated in the legend. Mambalgins are the only three-finger toxins to target ASIC channels and show no neurotoxic effect. (For

interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



drastically inhibit the ASIC currents present in spinal cordand hippocampal neurons, whereas PcTx1 was found toonly inhibit about 30% of the amplitude of these currents(Baron et al., 2008, 2002; Diochot et al., 2012). In sensoryneurons, mambalgins inhibit about 60% of ASIC currentsand PcTx1 about 40%, which has been attributed to theadditional inhibition by mambalgins of ASIC1b-containingchannels (Diochot et al., 2012).

APETx2 inhibits homomeric rat and human ASIC3channels (IC50 w37–63 nM for rat and w175 nM forhuman), and several heteromeric ASIC3-containing

channels: ASIC3 þ ASIC2b (IC50w117 nM), ASIC3 þ ASIC1b(IC50w0.9 mM) and ASIC3 þ ASIC1a (IC50w2 mM) (Anangiet al., 2010; Chagot et al., 2005; Diochot et al., 2004; Jensenet al., 2009). Upon acidification, ASIC3 channels generate aclassical transient and rapidly inactivating current and anadditional slowly activating, sustained current that doesnot inactivate as long as the pH remains acidic (Salinaset al., 2009; Waldmann et al., 1997a). APETx2 rapidly andreversibly inhibits the transient peak current and the sus-tained component evoked at pH 7.0 (Deval et al., 2011),which results from awindow current caused by the overlap

Table 1

Effects on ASIC currents of mambalgins, PcTx1, APETx2 and MitTx. Data obtained by application of the toxins at physiological pH 7.4 (unless specified) before

acidic pH stimulation on rat (r), chicken (c) and human (h) homomeric and heteromeric ASIC channels heterologously expressed in Xenopus oocytes or

mammalian cells. Inhibition (Inh.) or potentiation (Pot.) of the ASIC peak current activated by a drop in pH, or activation (Act.) in the absence of acid

stimulation is shown in red, purple and blue, respectively. The IC50 or EC50 values, and the concentration tested for potentiation are indicated when available.

No: no effect (the highest concentration tested is indicated). References: a (Diochot et al., 2012), b (Escoubas et al., 2000), c (Chen et al., 2005), d (Chen et al.,

2006), e (Diochot et al., 2004), f (Jensen et al., 2009), g (Anangi et al., 2010), h (Sherwood et al., 2011), i (Bohlen et al., 2011), j (Qadri et al., 2009), k (Samways

et al., 2009), l (Baconguis and Gouaux, 2012), m (Sherwood and Askwith, 2008), n (Hoagland et al., 2010), o (Saez et al., 2011), p (S. Diochot and M. Dauvois,

unpublished data using the Xenopus oocyte expression system; note that previous data in Diochot et al. (Diochot et al., 2012) have been obtained using

transfected COS-7 cells).

Channel Mamb-1

IC50

Mamb-2

IC50

Mamb-3

IC50

PcTx1

IC50

APETx2

IC50

MitTx

EC50

rASIC1aInh.

11p-55

a nM

Inh.

55 nMa

Inh.

17p nM

Inh.

0.4-3.7 nMb,c,o

Act.

9 nMi

rASIC1bInh.

44p-192

a nM

Inh.

44p-192

a nM

Inh.

44 nMp

Pot.

100 nMd

Act.

23 nMi

rASIC2aNo

up to 2 µMa

No

up to 3 µMa

No

up to 100 nMb

Pot.

at 75 nMi

rASIC3No

up to 2 µMa

No

up to 3 µMa

No

up to 100 nMb

Inh.

37-63 nMe,f,g

Act.

830 nMi

rASIC1a+ASIC2aInh.

246a-252

p nM

Inh.

252 nMp

Inh.

252 nMp

No

up to 10 nMb

Act.

at 75 nMi

rASIC1a+ASIC2bInh.

61 nMa

Inh.

3 nMh

rASIC1a+ASIC1bInh.

72 nMa

rASIC1a+ASIC3No

up to 2 µMa

No

up to 10 nMb

Inh.

2 µMe

No

up to 75 nMi

rASIC1b+ASIC3No

up to 2µMa

No

up to 3µMa

Inh.

900 nMe

rASIC2a+ASIC3No

up to 3µMe

No

up to 75 nMi

rASIC2b+ASIC3 Inh.

117 nMe

hASIC1a

(orthologue of

rASIC1a)

Inh.

127 nMa

Inh.

127 nMa

Pot.

at 60 nMn

Inh.

13 nM (pH6.2)j

venom (pH7.2)m

hASIC1b

(orthologue of

rASIC1b)

Pot.

at 60nMn

hASIC2a

(orthologue of

rASIC2a)

No

up to 670 nMa

No

up to 850 nMa

No

up to 25 nMj

hASIC1a+ASIC2aInh.

220 nMa

No

up to 100 nMj

hASIC3aInh.

175 nMe

cASIC1

Act.

189 nMl,k



between inactivation and activation of the peak current(Deval et al., 2008; Yagi et al., 2006), but the toxin does notaffect the sustained component evoked at pH 5.0 (Diochotet al., 2004). In sensory neurons, APETx2 inhibits the ASICcurrents that are insensitive to mambalgins (Diochot et al.,2012). APETx2 has been recently shown to also target tosome extent the Nav1.8 voltage-dependent Naþ channel(IC50 between 55 nM and 19 mM, depending of the study) aswell as NaV1.8-related currents in rat sensory neurons (IC50w2.6 mM) (Blanchard et al., 2012; Peigneur et al., 2012).APETx2 also inhibits Nav1.2 current (IC50 w114 nM) andslightly reduces the Nav1.6 current (17% inhibition by 1 mMtoxin) (Peigneur et al., 2012).

MitTx does not inhibit, but potently activates severalhomomeric and heteromeric ASIC channels (Bohlen et al.,2011; Bohlen and Julius, 2012). The most robust effectsare seen on homomeric rodent ASIC1a and ASIC1b currents(EC50 w9 and 23 nM, respectively), with a much lower ef-fect on ASIC3 current (EC50w830 nM). MitTx has onlyweakeffects on ASIC2a current when applied at physiological pH7.4, but strongly potentiates the acid-evoked current byshifting its activation curve towards less acidic pH. Anactivation of heteromeric ASIC1aþ ASIC2a channels, even ifweaker than the one of homomeric ASIC1a channels, is alsoreported. The effect of MitTx on native ASIC currents insensory trigeminal ganglion neurons seems to mainlydepend on ASIC1a-containing channels because this effectdisappears in neurons from ASIC1a-knockout mice (Bohlenet al., 2011).

3. Mechanisms of action and molecular basis of the

interaction of ASIC toxins with their target channels

Based on the crystal structure of chicken ASIC1 (theorthologue of rat ASIC1a), a model has been proposed foran ASIC subunit (Fig. 3A) resembling an upright forearmand clenched hand holding a ball (Jasti et al., 2007). Thetwo transmembrane domains (TM1 and TM2) form theforearm, the junction with the extracellular domain formsthe wrist, and the extracellular domain form the handdivided in palm, knuckle, finger, thumb and b-ball domains(Fig. 3B). Analysis of the interaction between PcTx1 andASIC1a channel has given a lot of information on thestructure and function of these channels, includingimportant information on channel open states throughrecent crystallization of a chicken ASIC1-PcTx1 complex(Baconguis and Gouaux, 2012) (Fig. 3C).

3.1. State-dependent binding of PcTx1 regulates the gating of

ASIC1a and ASIC1b channels

PcTx1 is a gating modifier that increases the apparentaffinity of rat ASIC1a channels for Hþ (Chen et al., 2005;Salinas et al., 2006). PcTx1 induces a shift of the pH-dependent inactivation curve of ASIC1a current towardsless acidic pH, which transfers almost all channels into aninactivated (desensitized) state at resting physiological pH7.4 (Fig. 4A, B). This effect is associated with a small shift ofthe activation curve towards less acidic pH, responsible fora minor stimulatory effect of PcTx1 observed with a pre-conditioning pH of 8.0, i.e., in the absence of pH-dependent

inactivation (Chen et al., 2005, and Fig. 4A, B). PcTx1 has asimilar effect on the human ASIC1a channel, but the inhi-bition is only observed at pH 7.2 with crude P. cambridgei

venom (Sherwood and Askwith, 2008). The differentbehaviour between rodent and human ASIC1a channels issupported by five divergent residues in the thumb domain,which render the pH-dependent inactivation of humanASIC1a channel less sensitive to pH. This shift prevents thepotent inhibitory effect of PcTx1 applied at preconditioningpH 7.4, the inhibition being only observed at lower pre-conditioning pH (Qadri et al., 2009; Sherwood and Askwith,2008).

PcTx1 promotes opening of rat ASIC1b channels atphysiological pH 7.4 (EC50 w100 nM) through a shift of itsactivation curve towards less acidic pH (Fig. 4C, D), whereas

Fig. 3. PcTx1 binds to the pH sensor of ASIC1a. A, Domain organization of a

single chicken ASIC1 subunit, showing the two transmembrane domains

(TM1 and TM2) flanking the large extracellular loop, with the same colour

code than in B. B, Schematic representation of the chicken ASIC1 structure

according to the model of a hand holding a ball (adapted from Jasti et al.,

2007), with two ASIC subunits out of three shown for clarity. Red arrows

indicate the domains proposed to be involved in channel gating by coupling

the extracellular domain with the transmembre domains TM1 and TM2 (see

text for details). C, 3D-structure of the chicken ASIC1-PcTx1 complex at pH

5.5 (side view; Baconguis and Gouaux, 2012). Solvent-accessible surface

representation of the channel, with subunits shown with different colours.

PcTx1 is shown in yellow. Inset: enlargement of the framed region where

PcTx1 binds into the acidic pocket of ASIC1. (For interpretation of the ref-

erences to colour in this figure legend, the reader is referred to the web

version of this article.)



Fig. 4. State-dependent effects of PcTx1 and mambalgin-1 on ASIC1a and ASIC1b channels. A, Schematic representation of the curves of pH-dependent activation

(right) and inactivation (left) of rat homomeric ASIC1a current, with or without toxins (protocols shown in inset). PcTx1 potently shifts the inactivation curve of

rat homomeric ASIC1a current, with also a slight effect on the activation curve. This leads to inhibition (red arrow) or stimulation (blue arrow) of the current. B,

Effect of PcTx1 on the different ASIC channels states, based on the Monod–Wyman–Changeux model for cooperativity in allostreric proteins (adapted from Chen

et al., 2006). Open (O), closed (C) and desensitized (D) states are indicated with different hypothetical levels of protonation depending on the extracellular pH

(indicated above) based on data from the literature (Chen et al., 2005, 2006; Diochot et al., 2012). Toxin is shown in italics when bound to a state of the channel,

and a frame indicates the state that is the most stabilized by binding of the toxin. Stimulatory or inhibitory effects of PcTx1 on the current are represented by blue

and red arrows, respectively (the width of the arrow is proportional to the potency of the effect). PcTx1 binds more tightly to the inactivated/desensitized state

and to a lesser extent the open state of rat ASIC1a, promoting either inhibition (PcTx1 applied at pH 7.4 followed by pH 5.0 stimulation), or stimulation in a range

of pH where negligible desensitization occurs (PcTx1 applied at pH 8.0 followed by pH 6.7 stimulation, or co-application of pH 7.1 þ PcTx1 from pH 7.4) (Chen

et al., 2005). C, PcTx1 shifts the activation curve of rat homomeric ASIC1b current, with almost no effect on the inactivation curve. D, Based on the model already

described in B, PcTx1 binds more tightly to the open state of rat ASIC1b, promoting opening (PcTx1 applied at pH 7.5 followed by pH 6.0 stimulation, or co-

application of pH 6.6 þ PcTx1 from pH 7.5). However, inhibition is also possible for instance when PcTx1 is applied at preconditioning pH 6.9 before a pulse

at pH 5.0 (Chen et al., 2006). E, PcTx1 (20 nM) is able to constitutively activate chicken ASIC1 channel at resting pH 7.4 (Samways et al., 2009) and to potentiate

the Hþ-activated current at pH 5.0. Red dashed line represents the current run-down. F, PcTx1 stabilizes the open state of chicken ASIC1, thus promoting

activation at pH 7.4 (O1 state) without desensitization (absent at pH 7.4). The peak current at pH 5.0 is also potentiated (O2 state). The absence of inhibitory effect

suggests that PcTx1 does not tightly bind to the desensitized state. The O1 and O2 states correspond to the open states stabilized by PcTx1 at high and low pH,

respectively, which have been described by Baconguis and Gouaux (2012). G, Mambalgin-1 induces a strong shift of the pH dependent activation of rat ASIC1a

towards more acidic pH, with only a minor effect on the steady-state inactivation that is shifted towards more alkaline pH. H, Mamb-1 binds more tightly to the

closed state and to a much lesser extent the inactivated/desensitized state of rat ASIC1a, promoting closure of the channel at any pH (for example, Mamb-1

applied at pH 7.4 followed by pH 5.0 stimulation). (For interpretation of the references to colour in this figure legend, the reader is referred to the web

version of this article.)


the inactivation curve is almost not affected. The differenceof PcTx1 affinity between the inactivated state of ASIC1aand ASIC1b channels depends on the adjacent upper part ofthe palm domain (see Fig. 3B) (Chen et al., 2006; Salinaset al., 2006). PcTx1 is able to constitutively activatechicken ASIC1 channel at resting pH 7.4 (Samways et al.,2009) and to potentiate the Hþ-activated current at pH5.0 (M. Salinas, personal communication, Fig. 4E) probablyby stabilizing the open state of the channel (Baconguis andGouaux, 2012) (Fig. 4F). The activating effect at pH 7.4 andthe potentiating effect at pH 5.0 are consistent with the twoopen-states stabilized by PcTx1 at high and low pHdescribed by Baconguis and Gouaux (2012). PcTx1 thusappears to have complex state-dependent effects on ASIC1aand ASIC1b channels that depend on the animal speciesand the corresponding pH-dependent properties of thechannel, and on the pH at which the toxin is applied. Thisleads to three different global effects of PcTx1: inhibition ofthe Hþ-gated ASIC current, potentiation of the Hþ-gatedASIC current, or activation of the ASIC current at physio-logical pH 7.4 (Table 1).

PcTx1 has a group of four basic residues (Lys25, Arg26,Arg27, Arg28), which forms a positive surface protrudingfrom the rest of the molecule (Escoubas et al., 2003) withthree aromatic residues (Trp7, Trp24, and Phe30) in thevicinity. This “basic-aromatic dyad”, equivalent to thosedescribed in scorpion or sea anemone toxins acting onvoltage-gated ion channels (Dauplais et al., 1997; Escoubaset al., 2003), has been proposed to be the functional surfaceof PcTx1 (Chagot et al., 2005; Escoubas et al., 2003).Moreover, the electrostatic anisotropy of PcTx1 generates adipole that emerges through the basic-aromatic dyad andcould play an orientating force within the electrostatic fieldof ASIC1a channel. The role of PcTx1 hydrophobic residuesTrp7 and Trp24 and basic residues Arg26, Arg27 and Arg28has been recently confirmed by the co-crystallization ofPcTx1 bound to the chicken ASIC1 channel (Baconguis andGouaux, 2012). The hydrophobic patch on PcTx1 seals thebasic cluster, enhancing the electrostatic interactions withacidic residues of the channel.

It has been proposed from data obtained by combining125I-PcTx1 binding and electrophysiological studies of ASICchimeras, that the thumb domain and the upper part of thepalm domain from an adjacent subunit are involved in thebinding site of PcTx1 on homomeric rat ASIC1a channel(Salinas et al., 2006) (Fig. 3B, C). Computer-aided moleculardocking analyses (Pietra, 2011; Qadri et al., 2009; Saez et al.,2011), and more especially the recent determination ofthe crystal structure of chicken ASIC1 bound to PcTx1(Baconguis and Gouaux, 2012; Dawson et al., 2012) haveconfirmed the role of these domains and brought new in-formation. Three PcTx1 molecules actually bind the ASIC1achannel at the interfaces between subunits, within theacidic pocket of the channel (Fig. 3B, C), a region (also called“pH sensor”) that is thought to play a key role in Hþ-dependent gating (Baconguis and Gouaux, 2012; Dawsonet al., 2012; Pietra, 2011; Qadri et al., 2009; Saez et al.,2011; Salinas et al., 2006; Sherwood et al., 2009).

In the upper part of the thumb domain of rat ASIC1a, thekey role of Asp349 and Phe350 (Asp350 and Phe351 inchicken ASIC1 or Asp351 and Phe352 in human ASIC1a) in

the interaction between PcTx1 and the channel has beenshown by mutagenesis (Salinas et al., 2006; Sherwoodet al., 2009) and from the crystal structure (Baconguisand Gouaux, 2012). The hydrophobic patch of PcTx1 (Trp7and Trp24) interacts with the thumb domain of the chan-nel, whereas the basic cluster (Arg26, Arg27 and Arg28)enters into the acidic pocket to form strong H-bonds(Baconguis and Gouaux, 2012).

Proton binding into the acidic pocket of the ASIC1achannel causes the disruption of carboxyl–carboxylatepairs and the release of coordinated Ca2þ (Gründer andXuanmao, 2010; Jasti et al., 2007). Both the Ca2þ-dependent binding of PcTx1 (Chen et al., 2005) and thehighly pH-dependent binding of 125I-PcTx1 (Salinas et al.,2006) support the possibility that arginine residues ofPcTx1 mimic the binding of protons to the ASIC1a acidicpocket which leads to steady-state inactivation (Saezet al., 2011).

The crystal structure of the PcTx1-bound chicken ASIC1channel has also allowed a better understanding of Hþ-gated inactivation of ASIC1a. From the open state, the lowerpalm domain and the wrist domain slightly rotate,strengthening the thumb and the palm domains of adja-cent subunits, which leads to the narrowing of the extra-cellular vestibule and induces a rearrangement of thetransmembrane domains responsible for the inactivation.Finger and thumb domains, which flank the palm domainand make major contributions to the acidic pocket, bindPcTx1 and presumably also Hþ, thereby modulatingmovements of the lower palm domain (Baconguis andGouaux, 2012).

3.2. Interaction between ASIC3 and APETx2

The interaction between APETx2 and ASIC3 has notbeen extensively studied yet. It is however possible tospeculate, based on the analysis of electrostatic anisot-ropy on its surface, that a particular patch of residues inAPETx2 could be involved in the interaction with ASICchannels. The presence of a “basic aromatic dyad” (Arg17,Arg31, Phe15, Tyr16 and Tyr32) and a basic-hydroxylcluster (Ser9, Lys10), which define a dipole momentemerging through the patch of basic residues (Chagotet al., 2005), may indeed suggest that APETx2 could alsobind to the acidic pocket to produce current inhibition via

a gating modifier mechanism involving an apparent shiftfor Hþ affinity. The importance of the Phe15 residue inAPETx2 for its inhibition of ASIC3 has been recentlydemonstrated (Anangi et al., 2012) by using an interestingEscherichia coli periplasmic expression system for theproduction of the peptide. Despite the fact that APETx2and PcTx1 display no sequence homology and no com-mon structural element, it is therefore possible that bothtoxins show similarities in their binding mechanism ontheir respective targets, i.e., ASIC3- and ASIC1a-containingchannels.

3.3. MitTx potentiates the activation of ASIC channels

The 3D-model of MitTx is very similar to b-bungar-otoxin (Kondo et al., 1978), and the co-crystallization of



b-bungarotoxin with its target, the nicotinic-acetylcholine-receptor, revealed that it deeply binds into theacetylcholine-binding domains at subunit interfaces via

extensive hydrophobic interactions, complemented byhydrogen bonding and electrostatic interactions. By ho-mology, it can be proposed that MitTx could also bind toASIC channels via its ligand-binding domains, i.e., the pHsensor. If this is true, one may expect some overlap be-tween the binding sites of MitTx and PcTx1 on ASIC1a,which is consistent with the fact that their effect are notadditive (Bohlen et al., 2011).

3.4. Mambalgins are gating modifier toxins that trap ASIC

channels in the closed state

Mambalgin-1 inhibits ASIC1a channel by inducing astrong shift of its pH-dependent activation towards moreacidic pH, trapping the channel in its closed state (Fig. 4G,H) (Diochot et al., 2012). Mambalgin-1 also induces amoderate shift of the steady-state inactivation towardsmore alkaline pH, stabilizing the channel in the inactivatedstate. The binding of mambalgin-1 thus appears to be state-dependent with a preference for the closed state andmarginally for the inactivated state. A 3D-model ofmambalgin-1 reveals a strong positive electrostatic po-tential, whichmay contribute, like for PcTx1 and APETx2, tofavour binding of the peptide to negatively charged regionsof ASIC channels (Diochot et al., 2012). Mambalgins are ableto inhibit homomeric ASIC1a but also ASIC1b channelssuggesting difference in the inhibitory mechanismcompared to PcTx1, which potentiates rather than inhibits

ASIC1b channel. The apparent lack of competition betweenPcTx1 and mambalgins in inhibiting the ASIC1a current(Diochot et al., 2012) is consistent with the fact that thesetoxins do not preferentially target the same state of thechannel (Fig. 4).

4. In vivo effects of ASIC-targeting toxins

In vivo exploration of the physiological and pathophys-iological roles of ASIC channels has been largely based onthe phenotypic analysis of knockout mice. However, avail-able ASIC-knockout mice are constitutive knockout, raisingthe possibility of the existence of compensatory mecha-nisms (related or not to ASICs), and conditional knockoutare not available yet. In this context, pharmacological ap-proaches in wild-type animals are of high potential andwell complement the knockout approach. Besides thenonspecific and/or subtype nonspecific small molecule in-hibitors of ASIC channels (like amiloride), specific ASIC-targeting peptides isolated from animal venoms arecertainly among the most interesting tools for the phar-macological exploration of the role of these channels in vivo

(Fig. 5).

4.1. Central injection of PcTx1 and mambalgins reveals the

role in pain of different ASIC1a-containing channels expressed

in neurons of the central nervous system

ASIC1a and ASIC2a/2b are largely expressed in centralneurons including spinal neurons that receive and modu-late primary afferent inputs, and neurons in brain areas

Fig. 5. In vivo effects of ASIC-targeting toxins. ASIC-cont., ASIC-containing; PIV, pressure-induced vasodilation, � and þ represent inhibition and stimulation of

ASIC channels, respectively.



associated with pain. Analysis of the functional propertiesof ASIC currents in cultured neurons, combined with theuse of ASIC-specific toxins, has shown that ASIC currents incentral neurons generally flow through a mixture ofASIC1a-containing channels, either homomeric ASIC1achannels or heteromeric ASIC1a þ ASIC2a orASIC1a þ ASIC2b channels (Baron et al., 2008, 2002;Sherwood et al., 2011). In spinal cord, their expression isup-regulated during peripheral inflammation and theyhave been proposed to play a role in the processing ofnoxious stimuli as well as in central sensitization associ-ated with hyperalgesia and allodynia in persistent pain(Duan et al., 2007;Wu et al., 2004). Intrathecal injections ofASIC-inhibitory toxins in the lumbar spinal cord of miceand rats clearly demonstrate a role for central ASIC1a-containing channels in the pain pathway, which was notanticipated from the phenotypic analysis of ASIC1a-knockout animals that have no major pain phenotype(Wemmie et al., 2003, 2002).

In mice, intrathecal (i.t.) and intracerebroventricular(i.c.v.) injections of PcTx1 were shown to induce a potentanalgesic effect in acute pain as well as inflammatory andneuropathic pain models (Mazzuca et al., 2007). Knock-down of ASIC1a after i.t. injection of antisense oligonucle-otides has a similar analgesic effect. This, together with thelack of effect of the toxin in ASIC1a-knockout animals (A.Baron, personal communication), supports the specificityof the PcTx1 effects in vivo. The analgesic effects of PcTx1are blocked by naloxone (an opioid receptor antagonist)and are absent in mice deficient for the preproenkephalingene. Blocking ASIC1a homomeric channels and probablyASIC1a þ ASIC2b channels (Sherwood et al., 2011) at thespinal and/or supra-spinal level thus activates the endog-enous opioid pathway and increases the level of Met-enkephalin in the cerebrospinal fluid (Mazzuca et al.,2007), which is associated with strong analgesic effects.Intrathecal injections of PcTx1 were also recently shown toprevent chronic abdominal pain in a rat model of irritablebowel syndrome (IBS) induced by butyrate (Matricon et al.,2011).

Central (i.t. and i.c.v.) injections of mambalgins in micewere shown to also induce potent analgesic effects in acuteas well as inflammatory pain (Diochot et al., 2012). Contraryto the effects of PcTx1, the effects of mambalgins areresistant to naloxone. The effect is completely lost inASIC1a-knockout mice demonstrating the essential impli-cation of ASIC1a-containing channels and the specificity ofmambalgins in vivo. Knockdown of ASIC2a after i.t. in-jections of siRNAs has a similar analgesic effect and reducessubsequent effect of the toxins, supporting the participa-tion of the ASIC2a subunit in the effect via heteromericASIC1a þ ASIC2a channels and providing the first evidenceof the involvement of ASIC2a in pain. Interestingly, thecentral analgesic effect of mambalgins can be as strong asmorphine but seems to produce less unwanted side effects(Diochot et al., 2012).

Different ASIC channels are therefore involved in twodifferent central neural pathways associated with paintransmission and/or modulation. Further studies areneeded to identify the cellular basis and neuronal networksinvolved in such pathways, but central ASIC channels

appear as promising therapeutic targets for novel analgesicdrugs.

4.2. Peripheral injections of APETx2, MitTx and mambalgins

reveal the role in pain and sensory perception of different ASIC

channels expressed in sensory neurons

4.2.1. APETx2 provides pain relief through inhibition of ASIC3-

containing channels

Peripheral injections of APETx2, combined with in vivo

gene silencing through i.t. injections of siRNAs, havedemonstrated the role of ASIC3 as a sensor of cutaneousacidic pain and post-operative pain as well as an inte-grator of molecular signals released during inflammationin rat, where it contributes to primary thermal hyper-algesia (Deval et al., 2010, 2011, 2008). Consistent withthese data, local peripheral application of APETx2 reducesmechanical hypersensitivity in a rat model of cutaneousinflammatory pain (Karczewski et al., 2010). This is ingood agreement with the increased expression of ASICs inrat sensory neurons induced by chronic inflammation ofthe hindpaw (Mamet et al., 2002, 2003; Voilley et al.,2001). The increase in ASIC3 expression, together withup-regulation of its activity by several components of the“inflammatory soup”, such as bradykinin, 5-HT (Devalet al., 2004), hypertonicity (Deval et al., 2008), arach-idonic acid (Allen and Attwell, 2002; Deval et al., 2008;Smith et al., 2007), nitric oxide (Cadiou et al., 2007), ATP(Birdsong et al., 2010) and polyamines (Li et al., 2010), maybe important for the sensitization of cutaneous noci-ceptors during inflammation. Interestingly and consistentwith the data in rat, a decrease in pH in the skin of humanvolunteers has been associated with non-adapting pain(Steen et al., 1995) and this cutaneous acid-induced painappears to be largely mediated by ASIC channels, espe-cially at moderate acid pH (pH> 6.0), since it is blocked byamiloride (Jones et al., 2004; McMahon and Jones, 2004;Ugawa et al., 2002).

ASICs are also expressed in sensory neurons thatinnervate muscle, joints and bone. ASIC3 is expressed inmore than 50% of small muscle afferents in rat (Devalet al., 2011; Molliver et al., 2005) and in more than 30%of sensory neurons innervating the knee joint in mouse(Ikeuchi et al., 2009). ASIC expression in sensory neuronsis increased in models of muscle inflammation (Walderet al., 2010) and acute arthritis (Ikeuchi et al., 2009)in mice. The combination of APETx2, amiloride andA-317567 (a small molecule non-discriminative inhibitorof ASICs), together with knockout and knockdown mice,has demonstrated an important role for ASIC3 in thegeneration of secondary mechanical hyperalgesia asso-ciated with central sensitization achieved in a mousemodel of non-inflammatory muscular pain induced byrepeated acid injections into the muscle (Price et al.,2001; Sluka et al., 2003) and in a mouse model of jointinflammation (Ikeuchi et al., 2008). Peripheral applica-tion of APETx2 reduces mechanical hypersensitivity ina similar model of non-inflammatory muscular painin rat (Karczewski et al., 2010). ASIC3 has also beeninvolved in the development of primary cutaneous me-chanical hyperalgesia induced by muscle inflammation



(Sluka et al., 2007; Walder et al., 2011). In a rat model ofostheoarthritis, continuous intra-articular injections ofAPETx2 reduced pain-related behaviour and secondarymechanical hyperalgesia, as well as the increase in ASIC3expression in knee joint afferent sensory neurons (Izumiet al., 2012).

4.2.2. APETx2 inhibits local vascular tone control through

blockade of ASIC3-containing channels

Blockade of ASICs by amiloride or APETx2 markedlyattenuates the exercise pressor reflex generated by con-tracting skeletal muscle in cats (Hayes et al., 2007;McCord et al., 2009) and rats (Tsuchimochi et al., 2011).The role of ASIC channels in the local vascular control issupported by the expression of ASIC3 in muscle metab-oreceptors, the sensory nerves that innervate musclearterioles and detect changes in muscle metabolism(Molliver et al., 2005).

Some studies of ASIC3-knockout mice have reportedsubtle alterations in normal cutaneous mechanicalsensitivity (Chen et al., 2002; Price et al., 2001), whileother studies were not able to show a significant contri-bution to mechanosensory function (Drew et al., 2004).By using APETx2, other pharmacological inhibitors of ASICchannels like amiloride and diclofenac (Voilley et al.,2001), and knockout mice, ASIC3 has been shown to bea neuronal sensor for the skin vasodilation response todirect pressure (a mechanism known as pressure inducedvasodilation or PIV) in both humans and rodents, and forskin protection against pressure ulcers in mice (Fromyet al., 2012).

4.2.3. MitTx evokes pain through activation of ASIC1a-

containing channels

Injection of MitTx in the mice hindpaw produces arobust pain-related behaviour (licking response) thatis decreased in ASIC1a-knockout mice but persists inASIC3-knockout animals, suggesting the involvement ofperipheral ASIC1a-containing channels in cutaneous pain(Bohlen et al., 2011). This is consistent with the effect ofMitTx on ASIC currents of cultured sensory trigeminalneurons, which seems to depend mainly on ASIC1a-containing channels because this effect disappears inneurons from ASIC1a-knockout, but not from ASIC3-knockout mice. On the other hand, subcutaneous in-jections of PcTx1 show no effect in acute, inflammatoryor post-operative pain models (Deval et al., 2008;Diochot et al., 2012; Duan et al., 2007; Mazzuca et al.,2007), which does not support a role for homomericASIC1a or heteromeric ASIC1a þ ASIC2b channels, butdoes not exclude a role for peripheral ASIC1a subunit inother heteromeric channels insensitive to PcTx1. It is alsonot clear at that point how pain produced by subcu-taneous injection of potent ASIC activators like MitTxrelates to pain detection triggered by a physiologicalstimulus, because ASIC1a channels inactivate over fewseconds when activated by Hþ (i.e., their physiologicalactivator), while the strong MitTx-evoked responses aremainly non-inactivating and therefore carry far morecurrent than would be produced even by a severeacidification.

4.2.4. Mambalgins provides pain relief through inhibition of

ASIC1b-containing channels

Subcutaneous injections of mambalgin-1 in the micehindpaw induces analgesic effects on acute heat pain andinflammatory heat hyperalgesia (Diochot et al., 2012).These effects are not involving ASIC1a, but ASIC1b-containing channels, as shown by the effect of ASIC1bsilencing with siRNAs that produces a similar analgesia andreduces subsequent effect of mambalgin-1, demonstratingan in vivo role for ASIC1b in pain detection.

4.3. Central injection of PcTx1 induces neuroprotective effects

through inhibition of ASIC1a-containing channels

In neurons of the central nervous system, loweringextracellular pH to the level commonly seen in ischaemicbrain activates ASIC1a currents, which results in increase inintracellular free Ca2þ concentration (Herrera et al., 2008;Samways et al., 2009; Xiong et al., 2004; Yermolaieva et al.,2004). Homomeric ASIC1a channels display some perme-ability to Ca2þ but the increase in intracellular Ca2þ seemsto primarily occur through the secondary activation ofvoltage-gated Ca2þ channels and Ca2þ release from intra-cellular stores (Zha et al., 2006). Activation of ASIC1a-containing channels during metabolic acidosis accompa-nying stroke has been proposed to contribute to neuronaldeath associated with glutamate-independent mecha-nisms of Ca2þ entry during ischaemia (acidotoxicity) (Gaoet al., 2005; Xiong et al., 2004).

PcTx1 shows neuroprotective effects on mouse culturedhippocampal CA1 neurons submitted to the OGD (oxygenand glucose deprivation) model of ischaemia (Gao et al.,2005) as well as on spinal motoneurons submitted toextracellular acidosis (Behan et al., 2013). These effects ofPcTx1 could involve inhibition of homomeric ASIC1achannels or heteromeric ASIC1a þ ASIC2b channels, asrecently proposed (Sherwood et al., 2011). The crudePcTx1-containing P. cambridgei venom also shows a neu-roprotective effect on cultured mice cortical neurons sub-mitted to acid injury (Xiong et al., 2004) as well as in theOGDmodel of ischaemia on cultured hippocampal neurons(Gao et al., 2005). It should be noted that in several in vivo

studies, the crude P. cambridgei venomwas used instead ofpure PcTx1 toxin, which only represents about 1% of thetotal protein content of the venom. It is not clear how othercomponents of the venom can affect the data, but resultsthat have been obtained with both pure toxin and thevenom are generally similar. Neurons from ASIC1a-knockout mice are also more resistant to acidotoxicityand ischaemia (Xiong et al., 2004) supporting theinvolvement of ASIC1a-containing channels.

In rat and mouse models of cerebral focal ischaemia,i.c.v. injection of P. cambridgei venom or PcTx1 as late as5 h after (venom) or 30 min before (PcTx1) a severe(60 min) transient middle cerebral artery occlusion(MCAO) reduces the infarct volume by more than 50% andthe protection persisted for at least 7 days (venom, noinformation for PcTx1) (Pignataro et al., 2007; Xiong et al.,2004). In the same MCAO model, ASIC1a-knockout micealso show 60–75% smaller infarct than wild-type mice(Duan et al., 2011; Xiong et al., 2004). In model of



ischaemia produced by asphyxia-induced cardiac arrestin pig, i.c.v. injection of P. cambridgei venom 20 minbefore hypoxia partially protects the neurons in putamen(Yang et al., 2011). Additive neuroprotection is obtainedafter pretreatment with an NMDA receptor antagonist(MK-801). P. cambridgei venom also shows some neuro-protective effects in the MPTP model of Parkinson’s dis-ease in mouse (Arias et al., 2008), as well as in a model ofmultiple sclerosis associated with axonal degeneration(Friese et al., 2007). In the later model, ASIC1a-knockoutmice present a reduced phenotypical deficit comparedto wild-type mice. Acid-induced axonal degeneration isalso reduced by P. cambridgei venom in an ex vivo modelof optic nerve and retinal explants (Friese et al., 2007).Interestingly, this neuroprotective effect is due to acombined action on neurons and oligodendrocytes thatare in charge of the axon myelinization and expressASIC1a-containing channels (Feldman et al., 2008; Vergoet al., 2011). Using a model of traumatic neuronal spinalcord injury in rats, Hu et al. (Hu et al., 2011) showed thati.t. injection of P. cambridgei venom or ASIC1a silencingwith antisense oligonucleotides reduces the lesion vol-ume and increases locomotor recovery 3 days after injury.This is correlated with a 40% reduction of neuronal deathin cultured spinal neurons.

Retina is a functionally distinct region of the centralnervous system that has been shown to express ASICchannels (Brockway et al., 2002) where they have beeninvolved in normal retinal activity (Ettaiche et al., 2006,2009, 2004). Intraocular (intravitreal) injection of PcTx1and antisense oligonucleotides against ASIC1a in rats showthat ASIC1a is a positive modulator of cone photo-transduction and adaptation (Ettaiche et al., 2006). Toxinswere not tested yet on in vivo pathological models oftransient global ischaemia, but PcTx1 reduces the in vitro

ischaemia-induced cell death of cultured rat retinal gan-glion cells (Tan et al., 2011) that have been shown to ex-press ASIC currents (Ettaiche et al., 2006). Ischaemic retinalganglion cell injury plays a role in a variety of retinal dis-eases such as diabetic retinopathy, hypertensive vasculardisease and glaucoma.

ASIC1a-containing channels can therefore representnovel therapeutic targets for ischaemic brain or retinainjury (Xiong et al., 2007), and PcTx1 a new therapeuticcandidate with neuroprotective properties (Wang and Xu,2011) in ischaemia and stroke therapy as well as retinaldiseases.

4.4. Fear reduction, antidepressant and anxiolytic effects of

central injection of PcTx1 through inhibition of ASIC1a-

containing channels in the amygdala

Expression of ASIC1a is particularly high in the amyg-dala, a brain region involved in fear, arousal and emotions(Wemmie et al., 2003). In this structure, ASIC1a acts as a pHsensor that contributes to the production of fear behaviourassociated with inhalation of carbon dioxide and acidosis(Ziemann et al., 2009). ASIC1a actually contributes to fearresponses to a variety of aversive stimuli (Maren, 2009),probably in part through a contribution to synaptic plas-ticity underlying the acquisition of conditioned fear

(Wemmie et al., 2002). In mice, i.c.v. injection of PcTx1-containing P. cambridgei venom reduces innate fear-related behaviour (Coryell et al., 2007). The venom isineffective in ASIC1a-knockout mice, which supports thespecificity of the venom effect towards ASIC1a. Interest-ingly, ASIC1a-knockout mice have reduced innate fear,whereas restoring ASIC1a expression in the basolateralamygdala rescues contextual fear conditioning (Coryellet al., 2008). Conversely, overexpression of ASIC1a intransgenic mice increases fear conditioning (Wemmieet al., 2004).

An i.c.v. injection of PcTx1 has antidepressant effects(Coryell et al., 2009). ASIC1a-knockout mice are resistantto depression, whereas PcTx1 is ineffective in these ani-mals, confirming the specificity of the toxin in vivo. P.cambridgei venom exerts the same antidepressant effectthan PcTx1. Restoring the expression of ASIC1a in theamygdala of ASIC1a-knockout mice with a viral vectoreliminates the antidepressant-like phenotype, suggestingthat ASIC1a in the amygdala plays a role in depression(Coryell et al., 2009).

Anxiolytic-like effects of PcTx1 have also beendescribed. In the stress-induced hyperthermia model, i.c.v.administration of P. cambridgei venom prevents stress-induced elevations in mice core body temperature(Dwyer et al., 2009). GABAergic mechanisms in the amyg-dalawere proposed to mediate these anxiolytic-like effects,but the precise pathways involved are not known.

4.5. PcTx1 inhibits vasoconstriction in vascular smooth

muscle cells through ASIC1a-containing channels

ASIC channels are expressed in vascular smooth musclecells (VSMC) from arteries where they could play a role inmechanotransduction of the myogenic response (pressure-induced adjustment of vascular tone) and VSMC migration(Drummond et al., 2008). ASIC currents recorded in freshlydissociated mice cerebral artery smooth muscle cells areenhanced by PcTx1 in 76% of cells, consistent withexpression of ASIC1b channels, and inhibited by PcTx1 in11% of cells, consistent with expression of ASIC1a-containing channels (Chung et al., 2010).

In rat pulmonary arteries, PcTx1 and ASIC1-silencingwith siRNA both reduce store-operated calcium entry inVSMCs, whereas others siRNAs against ASIC2 and ASIC3 arewithout effect (Jernigan et al., 2009). PcTx1 was also shownto reduce agonist-induced vasoconstriction and agonist-induced increase in intracellular Ca2þ (Jernigan et al.,2012). These results suggest that the inhibition of ASIC1a-containing channels rather than enhancement of ASIC1bcurrentsmay be involved in the effects of PcTx1 on agonist-induced vasoconstriction. Furthermore, the inhibition byPcTx1 of the hypoxia-induced enhanced store-operatedcalcium entry suggests that ASIC1a-containing channelscould be involved in the effect of chronic hypoxia onvascular tone. However, results obtained with ASIC2-knockout mice also suggest that a normal ASIC2 expres-sion could be required for a pressure-induced (myogenic)constriction of the mouse middle cerebral arteries, and thatASIC2 may also be involved in establishing the basalmyogenic tone (Gannon et al., 2008).



4.6. Improvement of glucose control by APETx2 through

inhibition of ASIC3-containing channels in adipocytes

Expression of ASIC3 subunit and recording of ASIC3current in adipose cells, as well as the lean phenotype ofASIC3-knockout mice related to a smaller size of adipo-cytes, were first reported by Huang et al. (2008). Interest-ingly, ASIC3-knockout mice also show enhanced insulinsensitivity and are protected against age-dependentglucose intolerance. Intraperitoneally injected APETx2 inaged wild-type mice produces the same effects, thus sup-porting a role for ASIC3 in age-dependent glucose intoler-ance and insulin resistance in adipocytes, which may relateto its capacity to sense lactate. Indeed, lactate can enhancethe opening of ASIC3 at near physiological pH (Immke andMcCleskey, 2001) and elevated basal level of lactate isconsistently found in diabetic subjects who also displaymarked insulin resistance (DiGirolamo et al., 1992).

4.7. Reduction of inflammation-induced apoptosis of

chondrocytes by PcTx1 and APETx2 through inhibition of

ASIC1a- and ASIC3-containing channels, respectively

ASIC3 has been shown to be expressed in joint afferentsensory neurons but also in chondrocytes and synovio-cytes, where they may be important in inflammatory anddegenerative joint disease (Ikeuchi et al., 2009; Kolkeret al., 2010). ASIC3-knockout mice does not develop sec-ondary hyperalgesia induced by carrageenan-inducedarthritis (Ikeuchi et al., 2008). In a rat model of ostheoar-thritis, the analgesic effects of APETx2 on ASIC3-containingchannels expressed in sensory neurons were com-plemented by a reduction of cartilage damage caused byinflammation (Izumi et al., 2012). Similar results have beenobtained with amiloride that protects articular chon-drocytes from acid-induced apoptosis (Hu et al., 2012),leading to chondroprotection in arthritis rats (Yuan et al.,2010a). PcTx1-containing P. cambridgei venom also signif-icantly inhibits the increase in intracellular Ca2þ and theacid-induced death of cultured rat articular chondrocytes(Yuan et al., 2010b), suggesting that the inhibition ofASIC1a-containing channels could also protectchondrocytes.

4.8. Other in vivo effects of ASIC-targeting toxins

In an in vivo model of kainate-induced epilepsy, i.c.v.injection of PcTx1 reduces both the seizure activity and theCA3 neuronal injury (Xiong et al., 2008), consistent withother studies showing an inhibition of seizures by ami-loride in rats andmice (Ali et al., 2006; Luszczki et al., 2009;N’Gouemo, 2008). This raises the possibility of a contribu-tion of ASIC channels, and ASIC1a-containing channels inparticular, to the generation and/or maintenance of seizure.However, other results have shown that i.c.v. injection of P.cambridgei venom or deletion of ASIC1a increased seizureseverity in mice, which has been associated with a pro-tecting role of ASIC1a-containing channels in seizuretermination by acidosis (a well-documented effect)through an increase in the inhibitory tone of inhibitoryinterneurons (Ziemann et al., 2008). Further studies will

certainly provide a better understanding of the differentcontributions of ASIC channels in epilepsy.

Recent data obtained in rats support the involvement ofASIC channels in central chemoreception and breathingcontrol. Injections of PcTx1 in the lateral hypothalamusinhibit ASIC1a channels in orexin neurons and the orexinaction on the medulla respiratory centre, which blocks theincrease in breathing frequency induced by extracellularacidosis (Song et al., 2012). ASIC-containing neurons arealso present in the nucleus of the solitary tract that projectto respiratory centres to increase breathing frequency inhypercapnic rats (Huda et al., 2012).

ASIC currents have been recorded from cultured neu-rons from rat inferior colliculus, partly flowing throughPcTx1-sensitive channels, suggesting a role for ASIC chan-nels in the central auditory system (Zhang et al., 2008).

Grade IV gliomas are the most common and mostaggressive of all brain tumours, exhibiting high rates ofproliferation and migration, often setting up secondary focidistant from the primary tumour. PcTx1 and ASIC1silencing inhibit migration and proliferation of glioma cells(Rooj et al., 2012).

Finally, PcTx1 (most probably P. cambridgei venomalthough not clearly specified by the authors) has beenrecently shown to block cortical spreading depression(CSD), a phenomenon associated with migraine withaura in human, in a rat model of cortical needle prick-induced CSD (Holland et al., 2012). Interestingly, a role inmigraine-related behaviour of peripheral ASIC3 channelsexpressed in dural afferents has also been proposed basedon the effect of amiloride (Yan et al., 2011).

4.9. Inhibitory ASIC-targeting toxins display no toxicity

ASIC-targeting inhibitory toxins (PcTx1, 0.46 mg i.t. or23 mg/kg; mambalgins, 2.2 mg i.t. and i.c.v. or 110 mg/kg;APETx2, 1.8 mg intraplantar; 0.9 mg intravenous; 2.7 mg i.t. or135 mg/kg) never produce excitotoxicity, spasms, convul-sions, motor paralysis, nor ataxia upon in vivo injections inmice. This is different from other spider, snake or seaanemone toxins like d-atracotoxins (3.2 mg s.c. or 160 mg/kg), dendrotoxins (8 mg/kg intracisternal), or ASII (2.5 mg/kgintracisternal) previously characterized as Nav activators orKv1 inhibitors, and responsible for high neurotoxicity(Mylecharane et al., 1989; Schweitz, 1984; Schweitz et al.,1990). PcTx1 and APETx2 are also not toxic in insects, i.e.,natural preys of the tarantula (intra-abdominal injection inthe cricket Acheta domestica of 470 and 450 ng/100 mg ofinsect body weight, respectively), whereas the scorpiontoxin AaHIT (80 ng/100 mg of insect body weight) provokesan intense but reversible paralysis in more than 50% of theanimals (S. Diochot and M. Dauvois, personal communi-cation). APETx2 is not toxic or lethal after in vivo injectionsin crustaceans (intramuscular injection in the shore crabCarcinus maenas) (Sanchez-Rodriguez, 1997), unlike othersea anemone NaV toxins previously described (ASII, AxI;Beress and Beress, 1975; Schweitz, 1984).

None of the ASIC inhibitory peptides is therefore toxicfor the natural preys of the venomous animal producing it(PcTx1 for cricket, APETx2 for crab, mambalgins formouse). If the role of pain-producing toxins like MitTx



could be to discourage threatening predators by triggeringa disorienting and memorable sensory experience, onecan wonder what could be the role in venom of ASIC-targeting analgesic toxins like PcTx1, APETx2 and mam-balgins. One possibility could be that these peptidesparticipate to hunting strategies, for instance to avoidalarming the bitten preys that could run away or fightback. It is interesting however to mention again thatPcTx1 does not block but activates chicken ASIC1a (seeTable 1). It is not known if PcTx1 can cause pain in birds,but as previously suggested (Gründer and Chen, 2010), thepresence of PcTx1 could be a defense strategy employedby the tarantula against potential predators. It would alsobe interesting to investigate if ASIC toxins are present inother tissues (i.e., outside the venom gland) to fulfilparticular physiological functions (for example as endog-enous analgesics), based on recent work suggesting pro-tein recruitment from the venom into non-toxic hostphysiological functions (Casewell et al., 2012).

5. Conclusion

There are now several venom toxins targeting ASICchannels with high levels of specificity both in vitro andin vivo, which can discriminate between subtypes ofASIC1- and ASIC3-containing channels. These peptidescan occasionally share some common targets (forexample, mambalgins and PcTx1) despite theircompletely different sequence and structure. Since thediscovery of the first ASIC-targeting toxin (PcTx1) in 2000,these peptides have been very important to better un-derstand the structure–function relationships of ASICchannels, the channel subtypes involved in native cur-rents and their implication in processes like nociception,mechanosensitivity, chemosensitivity, neuromodulationor neurodegeneration associated with a variety of situa-tions ranging from pain perception and modulation toneurological diseases including post-traumatic stress,anxiety, depression, stroke, epilepsy, neuroinflammatorydisorders and neurodegenerative diseases, as well asother emerging physiological situations in non-neuronalcells. ASIC channels generally need to be blocked to havefunctional benefits, but protective effects could also arisefrom channel activation like for instance in epilepsy.Whether ASIC channels are protective or deleterious inpathophysiological situations may probably depend onseveral parameters including in which cells they areactivated, to what extent and duration, and on the pres-ence of modulators of ASIC function.

ASIC channels appear therefore as important targets fordrug development in a variety of neurological and psychi-atric diseases, and probably also in other pathophysiolog-ical conditions outside the nervous system. ASIC-targetingpeptides isolated from animal venoms that specificallyblock this class of channels are therefore not only instru-mental as pharmacological tools to explore their functionbut also represent molecules of great potential therapeuticvalue. ASIC inhibitory peptides are also good examples ofthe high potential of non-toxic components of animalvenoms (Nirthanan et al., 2003; Utkin and Osipov, 2007).

Acknowledgements

We thank the Fondation pour la Recherche Medicale(FRM), the Agence Nationale de la Recherche (ANR), andthe Association Française contre les Myopathies (AFM) forfinancial support. We are grateful to D. Douguet for toxin3D-modelling, H. Schweitz for the sequence of mambalgin-3, L. Beress for crab toxicity informations, S. Cestele forproviding scorpion toxins used in the toxicity test withcrickets, and M. Dauvois, M. Christin, A. Delaunay and V.Friend for discussion and support.

Conflict of interest statement

The authors declare no conflict of interest.

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