14
Toxicon 49 (2007) 271–284 Peptides inhibitors of acid-sensing ion channels S. Diochot, M. Salinas, A. Baron, P. Escoubas, M. Lazdunski Institut de Pharmacologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique, Sophia-Antipolis, 660 Route des Lucioles, 06560 Valbonne, France Available online 4 October 2006 Abstract Acid-sensing ion channels (ASICs) channels are proton-gated cationic channels mainly expressed in central and peripheric nervous system and related to the epithelial amiloride-sensitive Na + channels and to the degenerin family of ion channels. ASICs comprise four proteins forming functional channel subunits (ASIC1a, ASIC1b, ASIC2a, and ASIC3) and two proteins (ASIC2b and ASIC4) without yet known activators. Functional channels are activated by external pH variations ranging from pH 0.5 6.8 to 4.0 and currents are characterized by either rapid kinetics of inactivation (ASIC1a, ASIC1b, ASIC3) or slow kinetics of inactivation (ASIC2a) and sometimes the presence of a plateau phase (ASIC3). ASIC1a and ASIC3, which are expressed in nociceptive neurons, have been implicated in inflammation and knockout mice studies support the role of ASIC3 in various pain processes. ASIC1a seems more related to synaptic plasticity, memory, learning and fear conditioning in the CNS. ASIC2a contributes to hearing in the cochlea, sour taste sensation, and visual transduction in the retina. The pharmacology of ASICs is limited to rather nonselective drugs such as amiloride, nonsteroid anti-inflammatory drugs, and neuropeptides. Recently, two peptides, PcTx1 and APETx2, isolated from a spider and a sea anemone, have been characterized as selective and high-affinity inhibitors for ASIC1a and ASIC3 channels, respectively. PcTx1 inhibits ASIC1a homomers with an affinity of 0.7 nM (IC 50 ) without any effect on ASIC1a containing heteromers and thus helped to characterize ASIC1a homomeric channels in peripheric and central neurons. PcTx1 acts as a gating modifier since it shifts the channel from the resting to an inactivated state by increasing its affinity for H + . APETx2 is less selective since it inhibits several ASIC3-containing channels (IC 50 from 63 nM to 2 mM) and to date its mode of action is unknown. Nevertheless, APETx2 structure is related to other sea anemone peptides, which act as gating modifiers on Nav and Kv channels. r 2006 Elsevier Ltd. All rights reserved. Keywords: ASIC; Toxin; PcTx1; APETx2; Spider; Sea anemone; Pain; Protons 1. Introduction Detection of acidosis by primary sensory neurons is an important way to inform the central nervous system (CNS) about damages occurring during tissue inflammation or hypoxia. The molecular basis of acid sensing was discovered when proton activated cationic currents were described in both central and peripheral nervous system more than 20 ARTICLE IN PRESS www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.09.026 Abbreviations: Kv, voltage-dependent potassium channels; Nav, voltage-dependent sodium channels; Cav, voltage-depen- dent calcium channels; PcTx1, psalmotoxin 1; NSAIDs, non- steroid anti-inflammatory drugs; DRG, dorsal root ganglia; CNS, central nervous system; ICK, inhibitor cystine knot; H-NMR, proton nuclear magnetic resonance Corresponding author. Tel.: +33 4 93 95 77 02/03; fax: +33 4 93 95 77 04. E-mail address: [email protected] (M. Lazdunski).

Peptides inhibitors of acid-sensing ion channels

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Page 1: Peptides inhibitors of acid-sensing ion channels

ARTICLE IN PRESS

0041-0101/$ - see

doi:10.1016/j.tox

Abbreviations

Nav, voltage-de

dent calcium ch

steroid anti-infla

CNS, central ne

H-NMR, proto�Correspondi

fax: +334 93 95

E-mail addre

Toxicon 49 (2007) 271–284

www.elsevier.com/locate/toxicon

Peptides inhibitors of acid-sensing ion channels

S. Diochot, M. Salinas, A. Baron, P. Escoubas, M. Lazdunski�

Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Sophia-Antipolis, 660 Route des Lucioles,

06560 Valbonne, France

Available online 4 October 2006

Abstract

Acid-sensing ion channels (ASICs) channels are proton-gated cationic channels mainly expressed in central and

peripheric nervous system and related to the epithelial amiloride-sensitive Na+ channels and to the degenerin family of ion

channels. ASICs comprise four proteins forming functional channel subunits (ASIC1a, ASIC1b, ASIC2a, and ASIC3) and

two proteins (ASIC2b and ASIC4) without yet known activators. Functional channels are activated by external pH

variations ranging from pH0.5 6.8 to 4.0 and currents are characterized by either rapid kinetics of inactivation (ASIC1a,

ASIC1b, ASIC3) or slow kinetics of inactivation (ASIC2a) and sometimes the presence of a plateau phase (ASIC3).

ASIC1a and ASIC3, which are expressed in nociceptive neurons, have been implicated in inflammation and knockout mice

studies support the role of ASIC3 in various pain processes. ASIC1a seems more related to synaptic plasticity, memory,

learning and fear conditioning in the CNS. ASIC2a contributes to hearing in the cochlea, sour taste sensation, and visual

transduction in the retina. The pharmacology of ASICs is limited to rather nonselective drugs such as amiloride,

nonsteroid anti-inflammatory drugs, and neuropeptides. Recently, two peptides, PcTx1 and APETx2, isolated from a

spider and a sea anemone, have been characterized as selective and high-affinity inhibitors for ASIC1a and ASIC3

channels, respectively. PcTx1 inhibits ASIC1a homomers with an affinity of 0.7 nM (IC50) without any effect on ASIC1a

containing heteromers and thus helped to characterize ASIC1a homomeric channels in peripheric and central neurons.

PcTx1 acts as a gating modifier since it shifts the channel from the resting to an inactivated state by increasing its affinity

for H+. APETx2 is less selective since it inhibits several ASIC3-containing channels (IC50 from 63 nM to 2mM) and to date

its mode of action is unknown. Nevertheless, APETx2 structure is related to other sea anemone peptides, which act as

gating modifiers on Nav and Kv channels.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: ASIC; Toxin; PcTx1; APETx2; Spider; Sea anemone; Pain; Protons

front matter r 2006 Elsevier Ltd. All rights reserved

icon.2006.09.026

: Kv, voltage-dependent potassium channels;

pendent sodium channels; Cav, voltage-depen-

annels; PcTx1, psalmotoxin 1; NSAIDs, non-

mmatory drugs; DRG, dorsal root ganglia;

rvous system; ICK, inhibitor cystine knot;

n nuclear magnetic resonance

ng author. Tel.: +334 93 95 77 02/03;

77 04.

ss: [email protected] (M. Lazdunski).

1. Introduction

Detection of acidosis by primary sensory neuronsis an important way to inform the central nervoussystem (CNS) about damages occurring duringtissue inflammation or hypoxia. The molecularbasis of acid sensing was discovered when protonactivated cationic currents were described in bothcentral and peripheral nervous system more than 20

.

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ARTICLE IN PRESSS. Diochot et al. / Toxicon 49 (2007) 271–284272

years ago (Akaike and Ueno, 1994; Alvarez de laRosa et al., 2002, 2003; Bevan and Yeats, 1991;Grantyn and Lux, 1988; Krishtal and Pidoplichko,1981; Ueno et al., 1992). Two types of protonactivated cationic channels are now well character-ized and classified as vanilloid receptors (VR1)(Caterina et al., 1997) and acid-sensing ion channels(ASICs). ASICs belong to the degenerin/epithelialsodium channel (Deg/ENaC) superfamily (Bianchiand Driscoll, 2002; Krishtal, 2003; Waldmann et al.,1996), and are related to the FMRFamide-gatedNa+ channel (FaNaC) identified from the inverte-brate nervous system which is the only ionotropicpeptide-gated channel (Lingueglia et al., 1995). Inmammalian organisms, six different proteins havenow been cloned: ASIC1a, ASIC1b, ASIC2a,ASIC2b, ASIC3 and ASIC4; these proteins areencoded by four genes. ASIC1b and ASIC2b aresplice variants of ASIC1a and ASIC2a. Subunits areable to arrange into homo- or heteromultimers toform functional channels (Babinski et al., 2000;Baron et al., 2001; Bassilana et al., 1997; Chu et al.,2004; Hesselager et al., 2004; Krishtal, 2003;Ugawa et al., 2003; Voilley et al., 2001; Waldmannet al., 1997b, 1999; Waldmann and Lazdunski,1998). These functional channels are activated byacidic pH to mediate a sodium-selective, amiloride-sensitive, cation current. Neither ASIC2b norASIC4 can form functional homomeric channels(Akopian et al., 2000; Grunder et al., 2000;Lingueglia et al., 1997), but ASIC2b has beenshown to associate with other subunits andmodulate their activity (Deval et al., 2004; Lingue-glia et al., 1997). The pharmacology of ASICchannels is restricted to nonselective drugswhich activate or inhibit the currents in high-concentration ranges. Two peptides recently iso-lated from spider and sea anemone venoms werecharacterized as selective and high-affinity blockersof ASIC1a and ASIC3 channels (Diochot et al.,2004; Escoubas et al., 2000). The spider toxin PcTx1proved to behave similar to a ‘‘gating modifiertoxin’’ by changing the H+ affinity of ASIC1a(Chen et al., 2005; Salinas et al., 2006). The seaanemone peptide APETx2 displays structuralelements common to other sea anemone toxinsknown to change the gating properties of Nav andKv channels (Chagot et al., 2005). These newlydiscovered peptides, which are potent blockers ofASIC channels, promise to be powerful tools tofurther elucidate the role of ASICs in neuronalexcitability and pain coding.

2. The ASIC channels: Structure, biophysiological

properties, and distribution

2.1. Structure

ASIC channels belong to a superfamily ofamiloride-sensitive cationic channels. They are wellconserved between rat, mouse and human. RatASIC isoforms share between 45% and 80% aminoacid sequence identities. Other ASIC channels havebeen characterized from toadfish, lamprey, sharkand zebrafish (Coric et al., 2005; Paukert et al.,2004). The mammalian degenerin (MDEG orASIC2a) channel was first cloned from rat andhuman brain (Price et al., 1996; Waldmann et al.,1996). It shares 67% sequence identity with ASIC1awhich was cloned from rat brain (Waldmann et al.,1997b). The splice variant ASIC2b was cloned frommouse and rat brain (Lingueglia et al., 1997) and isnot active by itself. Later the splice variant ofASIC1a, named ASIC1b was cloned from a ratDRG cDNA library and was found exclusively insensory neurons (Bassler et al., 2001; Chen et al.,1998). ASIC3 (previously called DRASIC) wascloned from rat and human dorsal root ganglianeurons (Babinski et al., 1999; de Weille et al., 1998;Ishibashi and Marumo, 1998; Waldmann et al.,1997a). More recently, ASIC4, which does notexpress any current by itself, was cloned from ratand human brain (Akopian et al., 2000; Grunderet al., 2000).

The ASIC membrane topology consists of twotransmembrane domains connected by a very largecysteine-rich extracellular loop with intracellularamino and carboxyl termini (Saugstad et al., 2004)(Fig. 3). In the N-terminal region, domains areinvolved in the selectivity and gating (Bassler et al.,2001; Coscoy et al., 1999). The first part of theextracellular loop after the transmembrane segmentM1 (residues 63–185 of ASIC1a, Fig. 3) is morevariable than the highly conserved second part(residues 186–432). His72 is important for pHsensing (Baron et al., 2001), and is closed to adomain related to the kinetics of desensitization(Coric et al., 2003). Near position 105 is a domainimportant for pH activation and steady-state in-activation (Babini et al., 2002). Lys133 correspondsto the high-affinity site for Zn2+ inhibition (Chu etal., 2004). Two domains (CRDI and CRDII) are richin cysteine residues, and both contain one His relatedto the Zn2+ coactivator effect on ASIC2a (Baronet al., 2001). Cys193 is potentially involved in a

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disulphide bridge with Cys93. The end of theextracellular loop, near the transmembrane segmentM1, contains the degenerin site (Champigny et al.,1998; Hong and Driscoll, 1994) and two amino acidscrucial for Ca2+ block of ASIC1a (Paukert et al.,2004). The amiloride site and the selectivity filter ofthe transmembrane segment M2 have been charac-terized (Ji et al., 2001; Kellenberger and Schild, 2002;Poet et al., 2001).

2.2. Sensitivity to pH, permeability, kinetics

The sensitivity to external H+, the activation andinactivation kinetics are key properties to define andcharacterize ASIC channels which have no voltagedependence. ASIC1a activates when the extracellularpH decreases rapidly from pH 7.4 to values belowpH 6.9 (pH0.5act 6.2–6.8) (Chen et al., 2006; de Weilleand Bassilana, 2001; Sutherland et al., 2001; Wald-mann et al., 1997b). ASIC1a generates a rapidlyactivating and inactivating current (t act: 5.8–13.7msand t inact: 1.2–4 s at pH 6) (Bassleret al., 2001; Sutherland et al., 2001). Its splice variantASIC1b generates currents characterized bypH0.5act ¼ 5.1–6.2 (Benson et al., 2002; Chen et al.,1998) with rapid kinetics: t act 9.9ms and t inact0.9–1.7 s at pH 6 (Bassler et al., 2001; Benson et al.,2002). The ASIC2a channel requires pH valuesbelow pH 6 for activation (pH0.5act 4.1–5) (Baronet al., 2001; Champigny et al., 1998; de Weille andBassilana, 2001). ASIC2a has distinct kineticsproperties compared to other ASICs since it activatesand desensitizes more slowly (t inact 3.3–5.5 s at pH5) (de Weille and Bassilana, 2001; Lingueglia et al.,1997). In the presence of external acidic pH between5.0 and 6.7, ASIC3 is the most rapidly activating andinactivating current with t act o5ms and t inactaround 0.4 s at pH 6 (Benson et al., 2002; de Weilleet al., 1998; Sutherland et al., 2001; Waldmann et al.,1997a). ASIC3 has biphasic inactivation kinetics witha sustained component (pH0.5act ¼ 3.5–4) that doesnot inactivate, a characteristic that can be associatedwith its role in pain that accompanies tissue acidosis(Babinski et al., 1999; Bevan and Yeats, 1991; Reehand Steen, 1996).

ASIC2b, which does not express current by itself,has been shown to associate with other ASICsubunits such as ASIC1a, ASIC2a and ASIC3 inheteromeric combinations. The resulting currentshave specific biophysical characteristics (Hesselageret al., 2004; Lingueglia et al., 1997). Heteromericassociations between subunits increase the functional

diversity by giving rise to ASIC currents withintermediate biophysical and pharmacological prop-erties (Babinski et al., 2000; Bassilana et al., 1997).

2.3. Tissue distribution

In situ hybridization, immunohistochemistry, andelectrophysiological studies allowed cellular locali-zation of ASICs in central and peripheric nervoussystem where they are almost exclusively expressed.Although the exact subunit composition of ASICsin native neurons has not always been determined,ASIC1a, ASIC2a and ASIC2b subunits have beenshown to be abundant in the brain (Alvarez de laRosa et al., 2003; Bassilana et al., 1997; Linguegliaet al., 1997; Price et al., 1996; Waldmann et al.,1996, 1997b; Wemmie et al., 2002) and in spinalcord neurons (Akopian et al., 2000; Babinski et al.,1999; Chen et al., 1998; Grunder et al., 2000; Wu etal., 2004). ASIC1a, ASIC2a and ASIC2b are highlyexpressed in cerebral cortex, hippocampus, cerebel-lum, striatum, habenula, amygdala and olfactorybulb (Baron et al., 2002; Bassilana et al., 1997;Garcia-Anoveros et al., 1997; Lingueglia et al.,1997; Waldmann et al., 1997b; Wemmie et al.,2003). In DRG, ASIC1a, ASIC1b, ASIC2b andASIC3 are predominant in small neurons (Alvarezde la Rosa et al., 2002; Bevan and Yeats, 1991; Chenet al., 1998; Krishtal and Pidoplichko, 1981;Lingueglia et al., 1997; Olson et al., 1998; Ugawaet al., 2005; Voilley et al., 2001) whereas ASIC2a ispresent in medium and large sensory neurons(Alvarez de la Rosa et al., 2002; Garcia-Anoveroset al., 2001). ASIC3 channels are found in largediameter mechanoreceptors and in small diameternociceptors (Price et al., 2001; Voilley et al., 2001).ASIC3 has also been found in nonneuronal tissuessuch as testis, lung and bone (Babinski et al., 1999;Ishibashi and Marumo, 1998; Jahr et al., 2005). Thepresence of ASIC1a, 2a, 2b, 3 and 4 mRNA hasbeen described in the rat retina (Ettaiche et al.,2004, 2006; Lilley et al., 2004). ASIC4, which cannotbe activated by an acidic drop of extracellular pH, isstrongly expressed in the pituitary gland and is alsodetected in brain, spinal cord, and inner ear(Grunder et al., 2000). Low levels of ASIC4 mRNAhave been found in DRG (Akopian et al., 2000).

3. Some of the roles of ASIC channels

The presence of ASICs in nociceptors suggeststhat these channels have a predominant role in pain

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associated with tissue acidosis (Bevan and Yeats,1991; Chen et al., 2002; Jones et al., 2004; Krishtal,2003; Krishtal and Pidoplichko, 1981; Olson et al.,1998; Price et al., 2001; Ugawa et al., 2002;Waldmann et al., 1996).

ASIC1a transcripts are mainly present in smallDRG neurons i.e. nociceptors, and proinflamma-tory mediators (like NGF, IL-1, serotonin, brady-kinin) are able to increase ASIC transcript levels invivo (Mamet et al., 2002; Voilley et al., 2001). Inaddition, nonsteroid anti-inflammatory drugs(NSAIDs) such as ibuprofen, flurbiprofen, oraspirin directly inhibit ASIC1a and ASIC3 currents,in their therapeutic concentration range, whichcould contribute to their analgesic effects (Voilleyet al., 2001). Indeed, NSAIDs abolish pain causedby inflammatory acidosis in human (Jones et al.,2004). The role of ASIC3 in pain is also supportedby the fact that ASIC3 null mutant mice display areduced latency in the onset of pain responses andmodified pain-related behaviors in a variety ofnociceptive tests: acetic acid-induced writhing test,high temperature thermal test, tail pressure test andmuscle pain (Chen et al., 2002; Mogil et al., 2005;Sluka et al., 2003). ASIC1a and ASIC2a areexpressed in dorsal root ganglion (DRG) andnodose ganglion neurons that innervate the sto-mach. In disorders of the proximal gastrointestinaltract, such as gastric ulcers, the biophysical proper-ties of these channels are modulated, suggesting acontribution to chemosensation and chemonocicep-tion (Sugiura et al., 2005). In sensory neurons ofcardiac afferents, ASIC currents fire action poten-tials in response to extracellular acidification thataccompanies myocardial ischemia. ASIC3 repre-sents the major sensor of myocardial acidity with anability to open at pH 7, a value reached in the firstfew minutes of a heart attack (Immke andMcCleskey, 2001).

In the CNS, null mutant studies have indicatedthat ASIC1 is involved in synaptic plasticity,learning/memory, and fear conditioning (Bianchiand Driscoll, 2002; Chu et al., 2004; Wemmie et al.,2002–2004). During ischemic brain injuries, such asischemia and stroke, acidosis activates ASIC1achannels, which are permeable to Ca2+, inducingneuronal damages (Gao et al., 2005; Yermolaievaet al., 2004). Knockout of the ASIC1a gene orASIC1a blockers such as amiloride and the toxinPcTx1 have been shown to protect the brain fromischemic injury (Benveniste and Dingledine, 2005;Xiong et al., 2004).

4. Pharmacology of ASIC channels

The mollusc neuropeptide FMRFamide, as wellas NPSF and NPFF mainly expressed in themammalian CNS and at high levels in the spinalcord (Devillers et al., 1995; Panula et al., 1999;Vilim et al., 1999), act as potentiators of currentsgenerated by ASIC3 homomeric and ASIC3/ASI-C2a heteromeric channels (Askwith et al., 2000;Catarsi et al., 2001; Deval et al., 2003) and to alower extent ASIC1a channels (Xie et al.,2002, 2003). These peptides increase the peakamplitude and slow inactivation of H+-gatedcurrents. The antidiuretic drug amiloride blocksthe transient current generated by ASIC1a,ASIC1b, ASIC2a and ASIC3 with a rather lowaffinity (IC50410 mM) (Chen et al., 1998; Wald-mann et al., 1996, 1997a, b). NSAIDs are also ableto inhibit ASICs (Voilley et al., 2001). However,amiloride, as well as NSAIDs, are not selective(Voilley et al., 2001).

Two peptides, the tarantula toxin PcTx1 and thesea anemone toxin APETx2 were recently isolatedand characterized for their potent and specificinhibitory properties against ASIC1a and ASIC3channels, respectively (Diochot et al., 2004; Escou-bas et al., 2000).

4.1. PcTx1 is a selective and high-affinity inhibitor of

ASIC1a

The first selective inhibitor of ASIC1a is Psalmo-toxin 1 (PcTx1). It was purified from the venom ofthe tarantula Psalmopoeus cambridgei (Escoubas etal., 2000). PcTx1 is a 40 amino acid peptidecrosslinked by three disulfide bridges, it has amolecular mass of 4689.40Da and basic properties(pI ¼ 10.38). As PcTx1 constitutes a minor compo-nent in the venom of P. cambridgei, synthetic andrecombinant toxins have been produced to investi-gate its structural and pharmacological properties(Escoubas et al., 2000, 2003). Native, recombinantand synthetic PcTx1 inhibit similarly homomericASIC1a channels in various cell expression systemswith a very high affinity (IC50 less than 1 nM)(Fig. 1(Aa), Table 1). This blockade occurs atpH 7.4 (closed state of the channel), it is reversibleand complete for 10 nM PcTx1 (Escoubas et al.,2000). The mode of action of PcTx1 has beendescribed in a recent work (Chen et al., 2005, 2006)and is closely related to that of ‘‘classical’’ gatingmodifier toxins. Those are known to modify the

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(a) (b) (c)

(a) (b) (c)

(A)

(B)

Fig. 1. Effects of PcTx1 and APETx2 on ASIC1a and ASIC3 currents. (Aa) Inhibition and reversibility of PcTx1 (10 nM) on ASIC1a

homomeric currents recorded in Xenopus oocytes, and stimulated by an acidic step to pH 6 every 30 s. Oocytes were maintained at

�60mV. (b) Absence of effect of PcTx1 (10 nM) on heteromeric ASIC1a+3 channels expressed in COS cells clamped at �60mV and

stimulated by a pH drop from 7.4 to 4. (c) Effect of PcTx1 (3 nM) on native currents recorded in DRG neurons in the whole cell voltage

clamp configuration. Neurons were clamped at �60mV and currents were activated by a pH drop from 7.4 to 6. (Ba) Inhibition and

reversibility of APETx2 (100 nM) on homomeric ASIC 3 currents recorded in Xenopus oocytes, and stimulated by an acidic step to pH 6

every 30 s. Oocytes were maintained at �50mV. (b) Effect of APETx2 (300 nM) on heteromeric ASIC2b+3 channels expressed on COS

cells clamped at �50mV and stimulated by a pH drop from 7.4 to 5. (c) Effect of APETx2 (1mM) on native ASIC3-like currents recorded

in rat sensory neurons in the whole cell voltage clamp configuration. Neurons were clamped at 50mV and currents were activated by a pH

drop from 7.4 to 5. This current corresponds to a population of PcTx1-resistant ASIC3 like currents, which represent 26.5% of the total

recorded DRG neurons. The current was maximally inhibited with 3mM APETx2 to 47% of the peak control amplitude.

S. Diochot et al. / Toxicon 49 (2007) 271–284 275

properties of voltage-dependent channels by shiftingtheir voltage dependence and in turn affecting theiropening and closing properties (Nicholson et al.,1996; Winterfield and Swartz, 2000). PcTx1, byincreasing the affinity of ASIC1a for H+, induces ashift of the steady-state desensitization curve (0.27pH units), and also of the activation curve, sufficientto transfer almost all the channel into a desensitizedstate at pH 7.4, rendering it unavailable foractivation (Chen et al., 2005). Therefore, wepropose to classify PcTx1 as a ‘‘gating modifier’’as it modifies the opening and closure properties ofASIC1a although by a mechanism different from

that of other previously described gating modifiertoxins. The inhibition of ASIC1a also depends onexternal Ca2+, which competes with PcTx1 bindingto the channel. PcTx1 is a selective blocker since itdoes not inhibit homomeric ASIC1b, ASIC2a orASIC3 nor heteromeric ASIC1a containing chan-nels (Fig. 1(Ab), Table 1) (Escoubas et al., 2000).PcTx1 has no effects on a variety of Kv, Nav andCav channels (Escoubas et al., 2000).

PcTx1 has been used as a tool to dissect thecontribution of homomeric ASIC1a currents tototal native proton gated currents in severalneuronal preparations. In small DRG neurons,

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Table 1

Activity of PcTx1 and APETx2 on homomeric and heteromeric

ASIC channels: activities of PcTx1 and APETx2 against different

ASIC channels combinations expressed in heterologous systems

and reported as IC50

Channel combination PcTx1 APETx2

ASIC1a 0.9 nM NA

ASIC1b No inhibitiona NA

ASIC2a NA NA

ASIC3 NA 63nM

ASIC1a+3 NA 2mMASIC1a+2a NA —

ASIC1b+3 — 0.9mMASIC2a+3 — NA

ASIC2b+3 — 117nM

Data are reported from previous studies (Chen et al., 2006;

Escoubas et al., 2000) for PcTx1 and (Diochot et al., 2004) for

APETx2. NA: no activity; —: not tested.aStimulation depending on experimental conditions (Chen

et al., 2006).

S. Diochot et al. / Toxicon 49 (2007) 271–284276

PcTx1 inhibits a subpopulation of rapidly inactivat-ing currents (E18% of neurons; t inact around 2 s)with an affinity (IC50) of 0.7 nM (Fig. 1(Ac))(Escoubas et al., 2000; Mamet et al., 2002). In theseneurons, proton-induced spike activities, whichmimic pain induction, can be suppressed by anapplication of PcTx1 (10 nM). In granule cells fromthe cerebellum, PcTx1 (10 nM) is able to completelyinhibit the H+-gated Na+ current, which argues forthe unique molecular presence of homomericASIC1a. In rat hippocampal cells, the use of PcTx1has indicated that ASIC1a homomers constituteE50% of the native H+-gated current in 80% ofneurons and 100% of the current in the remaining20% of neurons (Baron et al., 2002).

4.2. APETx2 inhibits ASIC3-containing channels

The sea anemone peptide APETx2 was purified asa major constituent of the venom of Anthopleura

elegantissima (Diochot et al., 2004). APETx2 is abasic (pI ¼ 9.59) peptide of 42 amino acids cross-linked by three disulfide bridges and has a molecularmass of 4561.10Da. APETx2 blocks homomericASIC3 channels expressed in oocytes or mammaliancells with an IC50 of 63nM (Table 1). This inhibitionis rapid and fully reversible in a few minutes(Fig. 1(Ba)). The ASIC3 current has two kineticcomponents, a transient peak and a sustainedplateau, when stimulated at pH 4. Only the transient

peak current is sensitive to APETx2. The selectivityof APETx2 is clear on homomeric currents since itdoes not block ASIC1a, ASIC1b and ASIC2achannels. Heteromeric ASIC3-containing channelsare inhibited but with less potency (IC504117nM)by the toxin (order of potency: ASIC2b+34ASIC1-b+34ASIC1a+3) (Fig. 1(Bb), Table 1). Theheteromer ASIC2a+3 is insensitive to high concen-trations of APETx2 (Diochot et al., 2004). Theselectivity of APETx2 for ASIC channels wasdemonstrated by its absence of effects on Kv orNav channels. In DRG neurons, APETx2 (3mM)inhibits 50% of an ASIC3-like current, mainlyconstituted by ASIC3 homomers and ASIC2b+3heteromers, which was recorded in 26.5% of theneurons (Fig. 1(Bc)) (Diochot et al., 2004).

5. Structure of peptide inhibitors of ASIC

Production of recombinant PcTx1 allowed thetwo-dimensional 1H-NMR determination of itsthree-dimensional structure in solution (PDB code1LMM) (Escoubas et al., 2003). The PcTx1structure consists of a compact disulfide-bondedcore from which three loops and N and C terminiemerge. The secondary structure is a three-strandedanti-parallel b-sheet, comprising residues 7–9, 21–24and 31–34 (Fig. 2(B)). Due to a marked electrostaticanisotropy, PcTx1 can be represented by a trun-cated cone with a dipole moment parallel to themain axis of the molecule. PcTx1 belongs to thefamily of ‘‘inhibitor cystine knot’’ (ICK) moleculeswhich comprise other spider and snail toxins actingon Cav, Kv and Nav channels (Adams et al., 1993;Craik et al., 2001; Norton, 1991; Norton andPallaghy, 1998; Swartz and MacKinnon, 1995).The cystine knot motif comprises a ring, formed bytwo disulfide bonds and their connecting backbonesegments, that is threaded by the third disulfidebond. This compact globular scaffold gives thesemolecules compact properties and a high resistanceto chemical reagents and is also well adapted forpresenting a variety of functional groups to diversebiological targets.

PcTx1 has an original primary sequence since ithas very few homologies with spider venom peptidesidentified to date (Fig. 2(A)). Only the cysteinedistribution is conserved between some spider andcone polypeptide toxins belonging to the ICKfamily (Escoubas and Rash, 2004; Narasimhanet al., 1994; Norton and Pallaghy, 1998). A greatnumber of spider toxins having this ICK fold have

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Fig. 2. Structure and active surfaces of PcTx1 and APETx2. (A) Sequence alignments of APETx2 with K+ channel modulators isolated

from sea anemone extracts. Black boxes indicate sequence identities and gray boxes sequence homologies with BDS-I, BDS-II (Anemonia

sulcata) and APETx1 (Anthopleura elegantissima). Sequence alignment of PcTx1 with other ion channel modulators isolated from spider

venoms: HpTx2 from Heteropoda venatoria, HaTx1 and o-GsTxSIA from Grammostola spatulata, SNX482 from Hysterocrates gigas and

PaTx1 from Phrixotrichus auratus. (B) Ribbon representation of the backbone folding of PcTx1 (ICK fold) and APETx2. Structures were

prepared with the program Rasmol v2.6 using the coordinates from the Protein DataBank database. (C) Models representing the active

surfaces of PcTx1 and APETx2. The residues are colored blue for basic residues, purple for aromatic residues, green for polar uncharged

residues and yellow for aliphatic residues.

S. Diochot et al. / Toxicon 49 (2007) 271–284 277

been characterized as gating modifiers since theymodify the voltage-dependent properties of currentsvia an external interaction with the voltage sensor(S4 segment) of ionic channels. Some of these spiderpeptides are o-agatoxins, and o-grammotoxin

which block Cav channels, hanatoxins, phrixotoxins,and scodratoxins, which inhibit Kv channels,m-agatoxins, and d-atracotoxins which act on Navchannels (Bourinet et al., 2001; Diochot et al., 1999;McDonough et al., 1997a, b; Nicholson et al., 1996;

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Skinner et al., 1989; Swartz and MacKinnon, 1995;Wang et al., 2004).

APETx2 structure (PDB code 1WXN) wasdetermined by two-dimensional 1H-NMR usingthe native toxin (Chagot et al., 2005). It consistsof a compact disulfide-bonded core composed of afour-stranded b-sheet from which a loop (15–27)and the N- and C termini emerge (Fig. 2(B)). Thefour strands include residues 3–6 (strand I), residues9–14 (strand II), residues 28–32 (strand III), andresidues 35–39 (strand IV). Strands I and II areconnected by a type II0-b turn (residues 6–9),strands III and IV are connected by a type I-b turn(residues 32–35) and strands II and III areconnected by the 15–27 loop. APETx2 is classifiedas a ‘‘disulfide rich all b-toxin’’ and belongs to thedefensin family which includes antimicrobial pep-tides from humans, and several toxins from snake,sea anemones and Platypus venom. APETx2 simi-larly to b-defensin like peptides is stabilized arounda three disulfide bonded hydrophobic core with a1–5, 2–4, 3–6 cysteine pairing. In the sea anemonegroup, several structures have been determined:Anthopleurin A from Anthopleura xanthogrammica,APETx1 from A. elegantissima, ATX and BDStoxins from Anemonia sulcata, Sh-I from Sticho-

dactyla helianthus. APETx2 displays the highestsequence homology (76%) with APETx1, a peptideisolated from the same venom which inhibitsHERG K+ channels in a voltage-dependent mannerby shifting the channel activation curve towardsmore depolarized potentials (Fig. 2(A)) (Diochotet al., 2003; Restano-Cassulini et al., 2006). Somesequence homologies are found with BDS peptides(57%) which block Kv3.4 channels, and with AP-A,AP-B, AP-C toxins (41–47%) which activate Navchannels as gating modifiers by shifting their voltagedependence to more negative potentials (Benzingeret al., 1998; Diochot et al., 2003; Khera andBlumenthal, 1996; Oliveira et al., 2004; Restano-Cassulini et al., 2006). All these toxins are organizedaround the same overall fold, despite their distinctpharmacological targets.

5.1. Functional surface of PcTx1

Spider toxins which act as gating modifiers ofvoltage-dependent channels display a functionalsurface defining a potential interaction surface withthe channel, composed of hydrophobic residuessurrounded by basic residues and sometimes acidicresidues anchoring the toxin to the target surface

through formation of salt bridges (Takahashi et al.,2000; Wang et al., 2004). The structure of PcTx1displays a considerable number of positivelycharged residues in the b-turn linking the twob-strands (loop4). A group of four residues (K25,R26, R27, R28) forms a positive surface protrudingfrom the rest of the molecule (Fig. 2(C)). Threearomatic residues (W7, W24, and F30) are found inthe vicinity of the basic residues and may beinvolved in the formation of functional dyadsequivalent to those described in scorpion or seaanemone toxins (Dauplais et al., 1997). Thus, theproposed functional surface of PcTx1 could berepresented by a positively charged patch formed byresidues K25–R28 associated to aromatic sidechains (F30, W7 and W24) and other surroundinghydrophobic or negatively charged residues (Escou-bas et al., 2003).

5.2. Functional surface of APETx2

Two clusters of amino acids may be important forthe interaction of APETx2 with the ASIC3 channel.One is constituted of residues A3, S5, N8 and K10in the first and second strand together with T39 andA41 in the C-terminus of the peptide which aremostly uncharged and hydrophobic residues sur-rounding a central Lys (K10). The second cluster iscomposed of residues Y16, R17, P18, R31 and T36,located after the first strand and in the third andfourth strands, which are mainly basic residues closeto a hydrophobic Y and a nonpolar core (Chagotet al., 2005) (Fig. 2(C)). The association of two basicand aromatic residues probably plays an importantrole in the interaction of the toxin with its receptorsurface, as is the case for several sea anemone andscorpion toxins which block Kv channels (Dauplaiset al., 1997).

PcTx1 and APETx2 do not display any sequencehomologies and their different folds do not predictany common structural elements. However, bothtoxins possess the same pattern of residues withsome degree of similarity in their exposed sidechains. In both PcTx1 and APETx2, a dipoleemerges through the basic/aromatic cluster, suggest-ing that they have the same electrostatic anisotropyrepartition and that their interaction with theirreceptor is mediated by the basic/aromatic cluster(K25, R26, R27, R28, W7, W24 and F30 for PcTx1and R17, R31, F15, Y16, Y32, and F33 forAPETx2). The fact that electrostatic anisotropycould play an orientating force within the electro-

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static field of the membrane receptor and that theorientation of the dipole could define the interactionsurface between a toxin and its target has beenextensively validated for ‘‘pore blocker’’ scorpiontoxins by site-directed mutagenesis studies. Thesame prediction method is now emerging withgating modifier toxins in numerous publicationsbut remains to be confirmed by mutagenesis studies.A second basic/hydroxyl cluster is present in the twotoxins and cannot be excluded as contributing totheir interaction with their receptor site on the ASICchannel (T37, T40 and K39 for PcTx1 and S9 andK10 for APETx2). The presence of a single acidicresidue is also common to PcTx1 and APETx2 (E19and D16 for PcTx1, D23 for APETx2).

6. Interaction of PcTX1 with ASIC1a

With both heterologously expressed and brainnative channels, 125I-PcTx1 toxin binding indicateda single family of sites with similar high affinities(Kd values of 213 and 371 pM, respectively). 125I-PcTx1 binding is not altered by drugs such asNSAIDs, amiloride or FMRFamide, that either

Fig. 3. PcTx1 binding site on ASIC1a channel and important structur

subunits of the tetrameric channel are shown to simplify representati

binding site on ASIC1a (CRDI and CRDII) are indicated in white. AS

but are necessary for PcTx1 to modify channel gating, thus leading to in

probably essential to regulate the positioning of CRDI and CRDII, th

post-M1 domain and the linker domain. The pre-M1 domain has been s

with the conserved motif His-Gly involved in the gating mechanism of E

of FaNaC was shown to be part of a large aqueous cavity, with the cha

close to the interior (Poet et al., 2001). In the M2 segment, some amino a

filter of ENaC (Kellenberger et al., 1999; Schild et al., 1997). The degene

affects a residue proposed to lie upstream of the M2 segment in or ne

1996). Close to this residue, two amino acids are crucial for Ca2+ blo

domain of ENaC contributes to ion permeation, suggesting that multip

inhibit (NSAIDs and amiloride) or modify theinactivation of the channel (FMRFamide). Thisobservation indicates that the PcTx1 binding siteconstitutes a new target for the development ofnovel and very specific ASIC blockers, thus leadingto the putative development of new analgesics.

6.1. PcTx1 does not bind directly to the ion pore but

acts as a gating modifier

The identification in the ASIC1a extracellularloop of structural elements involved in PcTx1binding has been done using both the 125I-PcTx1toxin and an electrophysiological approach, toanalyze independently the toxin binding parametersand the inhibitory effect on ASIC currents gener-ated by ASIC1a and a set of chimeras betweenASIC1a and ASICs uninhibited by PcTx1 (Salinaset al., 2006). In a first step, this study has suggestedthat the ion pore structure, constituted by trans-membrane M1 and M2 segments and intracellularpre-M1 and post-M2 regions (Fig. 3), are notdirectly involved in either the PcTx1 binding siteor in its mechanism of inhibition. The fact that

al determinants of ENaC/DEG/ASIC channel family. Only two

on in two dimensions. Domains directly involved in the PcTx1

IC1a domains in gray are not directly involved in the binding site

hibition. The ASIC1a linker domain between CRDI and CRDII is

e global shape being imposed by the disulfide bridge between the

hown to control ion permeability of ASIC1a (Bassler et al., 2001),

NaC (Grunder et al., 1999). The first transmembrane domain M1

rge selectivity filter in the outer vestibule and the ion gate located

cids were involved in the amiloride binding site and the selectivity

rin mutation (DEG), which causes a persistent channel activation,

ar the pore of ASICs (Champigny et al., 1998; Waldmann et al.,

ck of ASIC1a (Paukert et al., 2004). The intracellular post-M2

le sites contribute to ion selectivity (Ji et al., 2001).

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amiloride, a known pore blocker of ENaC/DEG/ASIC channels (Schild et al., 1997), does not inhibitPcTx1 binding also supports this conclusion. More-over, a physical occlusion of the ion pore is notcompatible with the PcTx1-induced stimulatoryeffect observed with some ASIC1a chimeras (Sali-nas et al., 2006) and ASIC1b (Chen et al., 2006). Ina second step, it has been shown that (i) PcTx1binds principally on both cysteine-rich domainsI and II (CRDI and CRDII) of the extracellularloop; (ii) the post-M1 and pre-M2 regions, althoughnot involved in the binding site, are crucial for theability of PcTx1 to inhibit ASIC1a current bymodification of channel gating; (iii) the linkerdomain between CRDI and CRDII is importantfor their correct spatial positioning to form thePcTx1 binding site.

The model in Fig. 3 is consistent with thehypothesis that PcTx1 acts as a gating modifier onASIC1a by shifting the channel from its restingtowards its inactivated state through an increase ofits apparent affinity for protons, the natural ligandsof ASIC channels (Chen et al., 2005, 2006). Theinteraction of ASIC1a with PcTx1 is state depen-dent (Chen et al., 2006). The peptide binds mosttightly to the open and the inactivated states ofASIC1a.

7. Conclusion

ASIC channels are key channels present in a largevariety of neurons and implicated in a variety ofsensory modalities, including pain perception parti-cularly during inflammation or ischemia, possiblymechanotransduction and hearing (Drew et al.,2004; Hildebrand et al., 2004; Roza et al., 2004),visual transduction (Ettaiche et al., 2004; Lilleyet al., 2004), but also neuronal central processessuch as learning, memory, fear conditioning andsynaptic plasticity (Bianchi and Driscoll, 2002; Chuet al., 2004; Wemmie et al., 2002–2004). PcTx1 andAPETx2 represent the first high-affinity and selec-tive natural inhibitors for ASIC channels, which areimportant sensors of external pH variations inneurons. PcTx1 was demonstrated to have anoriginal mode of action on ASIC1a channels,classing it in the gating modifier category. To date,a large number of gating modifier toxins have beenisolated from scorpion, spider and sea anemonevenoms, acting on Nav, Cav and Kv channels.PcTx1, by shifting the pH dependence (gating) ofASIC1a channels, is the first member of a new type

of channel modulator. The mode of action ofAPETx2, which presents structural homologies withother sea anemone gating modifier toxins, remainsto be determined and compared with that of PcTx1.The effects of both PcTx1 and APETx2 inphysiological experiments using diverse models ofpain, inflammation, mechanosensitivity and adap-tative behaviors (fear conditioning, memory, learn-ing) will now help to better understand the functionof ASIC channels. PcTx1 was particularly interest-ing in showing the sensing role of ASIC1a in thevisual system (Ettaiche et al., 2006). Neutralizationof ASIC1a function by PcTx1 decreases significantlyand reversibly the photopic waves and oscillatorypotentials.

Acknowledgments

We thank the Association Franc-aise contre lesMyopathies (AFM), the Fondation pour laRecherche Medicale (FRM), and the AgenceNationale de la Recherche (ANR) for financialsupport.

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