a novel toxin variant from the Androctonus australis scorpion...

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Subtype-selective activation of Kv7 channels by AaTXKβ(2-64),

a novel toxin variant from the Androctonus australis scorpion venom

Zied Landoulsi, Francesco Miceli, Angelo Palmese, Angela Amoresano, Gennaro Marino,

Mohamed El Ayeb, Maurizio Taglialatela, and Rym Benkhalifa

Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis, Université Tunis-El

Manar, Tunis-Belvédère, Tunisia (Z.L., M.E.A., and R.B.); Div. Pharmacology, Dept. of

Neuroscience, University of Naples Federico II, Naples, Italy (F.M and M.T.); Dept. of Chemical

Sciences, University of Naples Federico II, Naples, Italy (A.P., A.A., and G.M.); Dept. of Medicine

and Health Science, University of Molise, Campobasso, Italy (M.T.); Unidad de Biofísica, Consejo

Superior de Investigaciones Cientificas – Universidad del Pais Vasco, Leioa, Spain (M.T.)

Molecular Pharmacology Fast Forward. Published on September 9, 2013 as doi:10.1124/mol.113.088971

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 9, 2013 as DOI: 10.1124/mol.113.088971

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Running title: A novel toxin activating Kv7 channel subtypes

Corresponding Authors: Maurizio Taglialatela Department of Medicine and Health Sciences University of Molise Via De Sanctis, 86100 – Campobasso, ITALY

Tel. (+39) 0874-404851; Fax: (+39) 0874-404778 Email: [email protected] Rym Benkhalifa Laboratoire et Molécules Thérapeutiques Group of Cellular Electrophysiology Institut Pasteur de Tunis BP74 - 1002 Tunis, TUNISIA Tel. (+21) 671843755 ext. 490; Fax (+21) 671791833 Email: [email protected]

Number of text pages: 30

Number of tables: 0

Number of Figures: 8

Number of References: 56

Word Count Abstract: 198

Word Count Introduction: 578

Word Count Discussion: 1541

Abbreviations: αKTx’s, short chain potassium channel toxins; β-KTx’s, long chain potassium

channel toxins; Cs-α/β, cysteine-stabilized α-helix/β-sheet; Aa, Androctonus Australis; KTX,

kaliotoxin; KTX2, kaliotoxin-2; TFA, Trifluoroacetic acid; ACN, acetonitrile; MBS, Modified

Barth’s Solution; CHO cells, Chinese hamster ovary cells; DMEM, Dulbecco’s modified Eagle’s

medium; DTT, dithiothreitol; SIM, Single Ion Monitoring; TEA, tetraethylammonium; MarTx,

martentoxin; DkTx, double-knot toxin; TRPV1, transient receptor potential vanilloid-1.


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Abstract

Kv7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair

cells, skeletal and smooth muscle cells. Openers of Kv7.4 channels have been suggested to improve

hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and

neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of

peptides active on K+ channels; therefore, a combined purification/screening procedure has been

optimized in the present work to identify specific modulator(s) of Kv7.4 channels from the venom

of the North African scorpion Androctonus australis (Aa). We report the isolation and functional

characterization of AaTXKβ(2-64), a novel variant of AaTXKβ(1-64) in an HPLC fraction from the Aa

venom (named P8), which acted as the first peptide activator of Kv7.4 channels. In particular, in

both Xenopus oocytes and mammalian CHO cells, AaTXKβ(2-64), but not AaTXKβ(1-64),

hyperpolarized the threshold voltage of current activation and increased the maximal currents of

heterologoulsy-expressed Kv7.4 channels. AaTXKβ(2-64) also activated Kv7.3, Kv7.2/3, and Kv7.5/3

channels, whereas homomeric Kv1.1, Kv7.1 and Kv7.2 channels were unaffected. We anticipate that

these results might prove useful to unravel novel biological roles of AaTXKβ(2-64)-sensitive Kv7

channels, and to develop novel pharmacological tools allowing subtype-selective targeting of Kv7

channels.


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Introduction

The Kv7 subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5)

each playing distinct pathophysiological roles in various cell types (Soldovieri et al., 2011). Kv7.1

subunits represent the molecular basis of IKs, a cardiac current involved in action potential

repolarization, whereas Kv7.2 and Kv7.3 genes have a predominant neuronal expression.

Heteromeric assembly of Kv7.2 and Kv7.3 subunits underlies the M-current (IKM), a subthreshold

current inhibiting repetitive action potential firing. Kv7.4 channels are expressed in central auditory

pathways and in inner ear sensory hair cells (Kubisch et al., 1999), in skeletal muscle cells during

myotube formation (Iannotti et al., 2013), and in smooth muscle cells of the gastrointestinal tract

(Jepps et al., 2009; Ipavec et al., 2011). In blood vessels, Kv7.4 channels contribute to vascular

reactivity to vasopressors and vasodilators (Yeung et al., 2007; Mani and Byron, 2011; Martelli et

al., 2013), and down-regulation of Kv7.4 contributes to primary and secondary hypertension (Jepps

et al., 2011). Kv7.5 subunits are expressed in neurons, showing a distribution largely overlapping

those of Kv7.2 and Kv7.3, and are thought to contribute to IKM heterogeneity (Schroeder et al.,

2000); Kv7.5 transcripts and/or proteins have been also identified in skeletal and smooth muscle

cells (Soldovieri et al., 2011).

Activation of K+ currents mediated by distinct Kv7 subunit combinations is currently

regarded as an effective pharmacological strategy against several human diseases, with cardiac

Kv7.1 channels being potential targets for antiarrhythmics (Tamargo et al., 2004), and neuronal

Kv7.2 and Kv7.3 channels for antiepileptics and analgesics (Miceli et al., 2008). Kv7.4-selective

openers may improve hearing loss (Leitner et al., 2012), systemic or pulmonary arterial

hypertension (Mani and Byron, 2011), urinary incontinence (Rode et al., 2010), gastrointestinal

(Jepps et al., 2009; Ipavec et al., 2011) and neuropsychiatric (Hansen et al., 2008) diseases, and

skeletal muscle disorders (Iannotti e al., 2010; Iannotti et al., 2013). However, most of available

synthetic compounds discriminated poorly among Kv7 channels formed by distinct combinations of

Kv7.2-5 subunits (Xiong et al., 2008). Therefore, compounds showing increased Kv7 subtype-

selectivity, beside representing important probes to elucidate the molecular pathophysiology of Kv7

channels at distinct sites, may offer additional therapeutic benefits (Wickenden et al., 2008).

Scorpion venoms are a large source of peptides active on K+ channels (KTx’s) (Rodriguez

de la Vega and Possani, 2004). According to their size, KTx’s can be classified into short chain

toxins (αKTx’s), consisting of 23-42 amino acids peptides stabilized by 3 or 4 disulfide bridges

(Tytgat et al., 1999), and long chain toxins (β-KTx’s) of 45-75 residues stabilized by 3 disulfide

bridges (Diego-Garcia et al., 2007). Two structurally distinct domains can be recognized in β-


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KTx’s: a putatively α-helical N terminus, and a cysteine-rich C-terminal domain containing a

common structural motif named Cs-α/β for cysteine-stabilized α-helix/β-sheet (Zhu et al., 2004).

Remarkably, venom peptides mostly act as K+ channel blockers, whereas naturally-occurring K+

channel activators have been rarely found.

Several KTx’s have been described in Androctonus scorpion species. These include αKTx’s,

such as kaliotoxin (KTX) (Crest et al., 1992) and kaliotoxin-2 (KTX2) (Laraba-Djebari et al.,

1994), and, among βKTx’s, AaTXKβ (Legros et al., 1998). Therefore, the aim of the present work

has been to investigate the from Androctonus Australis (Aa) scorpion venom as a potential source of

novel molecular entities active on Kv7 channels. We report the biochemical and electrophysiological

characterization of AaTXKβ(2-64), a novel molecular variant of AaTXKβ, as the first peptide

activator of Kv7.4 channels, also acting as a subtype-selective activator of channels formed by

Kv7.3, Kv7.2/3, Kv7.5/3 subunits.


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Materials and Methods

Materials

Trypsin, α-cyano-4-hydroxycinnamic acid, dithiothreitol and NH4HCO3 were from Sigma

(St. Louis, MO, USA). Trifluoroacetic acid (TFA)-HPLC grade was from Carlo Erba (Milan, Italy).

Retigabine (N-(2-Amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester) was from

Valeant Pharmaceuticals (Aliso Viejo, CA, USA). All other reagents and solvents were of the

highest purity available (J.T. Baker, Phillipsburg, NJ, USA).

Scorpion venom

Venom of Androctonus australis (Aa) was collected from Beni Khedach (Tunisia) by the

veterinarian service of the Pasteur Institute of Tunis and kept frozen at -20 °C in its crude form until

use.

Purification procedures

Crude venom was dissolved in water and loaded on Sephadex G50 gel filtration

chromatography columns (2 x K26/100; Pharmacia). Columns were equilibrated and eluted with 0.1

M acetic acid buffer, pH 4.7. After freeze-drying, the resolved fractions were stored at -20°C until

use. The elution profile of M2Aa was eventually collected in 4 sub-fractions (M2a, M2b, M2c, and

M2d). HPLC purification of M2c was performed using a C8 reversed-phase HPLC column (5µm,

4.6x250mm, Beckman Coulter, Brea, CA, USA) equipped with a Beckman series 125 pump and

Beckman diode array detector set at 214 and 280 nm. Elution was controlled by means of the

GOLD software (32 Karat version 7.0, Beckman Coulter), at a rate of 0.8 ml/min using a linear

gradient (30 min) from 20 to 50% of solution B (0.1% TFA in acetonitrile (ACN)) in solution A

(0.1% TFA in water). Protein concentration was measured by the Bradford protein assay

(Fermentas, Cornaredo, Italy) using bovine serum albumin as a standard.

cRNA transcription

The cDNA encoding for human Kv7.4 was cloned in the Xenopus oocyte expression vector

pTLN. After linearization with HpaI, capped cRNA was transcribed in vitro using the SP6

mMessage mMachine kit (Ambion, Foster City, CA, USA).

Expression in Xenopus laevis oocytes


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Xenopus leavis females were anesthetized with 0.17 % solution of 3-aminobenzoic acid

ethyl ester methanesulfonate salt (Sigma-Aldrich, Saint-Quentin Fallavier, France), and parts of

ovaries were surgically removed from the abdominal cavity and bathed in sterile Modified Barth’s

Solution (MBS) of the following composition (in mM): 88 NaCl, 1 KCl, 0.4 MgCl2, 2.4 NaHCO3,

0.8 MgSO4, 10 HEPES, 2.4 CaCl2, and 0.3 Ca(NO3)2, pH 7.4. Xenopus oocytes were then

defolliculated enzymatically by incubation for 2h in sterile MBS containing 2 mg/ml collagenase

(type A and type B; Roche, Indianapolis, IN, USA), followed by 3-4 washes in MBS. Stage V and

VI oocytes were injected with 50 nl of mRNA (10 ng/oocyte) using an automatic microinjector

(Nanoject Drummond, USA). Oocytes were kept at 18°C in sterile MBS supplemented with 0.1

mM gentamycin until electrophysiological experiments were performed.

Cell culture, transient transfection, and plasmids

Channel subunits were expressed in Chinese hamster ovary (CHO) cells by transient

transfection, using plasmids containing cDNAs encoding human Kv7.1, Kv7.2, Kv7.3, Kv7.4, Kv7.5,

and Kv1.1, all cloned in pcDNA3.1 (Life Technologies, Monza, Italy). Two additional plasmids

encoding for human Kv7.5 subunits were also tested: Kv7.5 in pcDNA3zeo, and Kv7.5 in a pIRES2-

DsRed-Express vector from Open Biosystems (Thermo Scientific, Lafayette, CO, USA). According

to the experimental protocol, these plasmids were expressed individually or in combination,

together with a plasmid expressing enhanced green fluorescent protein (Clontech, Palo Alto, CA,

USA) used as a transfection marker. Total cDNA in the transfection mixture was kept at 4 µg. CHO

cells were grown in 100 mm plastic Petri dishes in Dulbecco’s modified Eagle’s medium (DMEM)

containing 10% fetal bovine serum, non-essential amino acids (0.1 mM), penicillin (50 U/ml), and

streptomycin (50 µg/ml) in a humidified atmosphere at 37°C with 5% CO2. 24h before transfection,

cells were plated on glass coverslips (Carolina Biological Supply Company, Burlington, NC, USA)

coated with poly-L-lysine and transfected on the next day with the appropriate cDNAs using

Lipofectamine 2000 (Life Technologies, Monza, Italy), according to the manufacturer’s protocol.

Electrophysiological experiments were performed 24 hours after transfection.

Two microelectrode voltage clamp

4-6 days after mRNA injection, two-microelectrode voltage-clamp measurements in

Xenopus oocytes were performed at room temperature (22–24°C) using a Geneclamp 500B

amplifier combined with Digidata 1322A (Molecular Devices, Foster City, CA). Micropipettes were

pulled from borosilicate glass capillaries on a Flaming/Brown type pipette puller (P-97, Sutter

instruments, USA) and had a tip resistance of 1-2 MΩ when filled with 3 M KCl. Data was filtered


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at 1 KHz and voltage step protocols and current analysis were performed with pCLAMP 8.2

software (Molecular Devices, Foster City, CA). During the recordings, oocytes were perfused with

ND96 solution containing (in mM): 96 NaCl, 4 KCl, 1 MgC12, 1.8 CaC12, 5 HEPES; pH 7.6, in a

small chamber (2 ml volume). The perfusion system was controlled by a Manifold Solution

Changer (MSC-200, Bio-Logic, Grenoble, FR). From a holding potential of -80 mV, Kv7.4 currents

were activated with voltage pulses from -100 to +40 mV in 10 mV steps, followed by a constant

pulse to -120 mV of 300 ms duration.

Patch clamp

Currents from CHO cells were recorded at room temperature (20–22°C) using the whole-

cell configuration of the patch-clamp technique, with glass micropipettes of 3–5 MΩ resistance. No

compensation was performed for pipette resistance and cell capacitance. During the recording,

constant perfusion of extracellular solution was maintained. The extracellular solution contained (in

mM): 138 NaCl, 2 CaCl2, 5.4 KCl, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. The

pipette (intracellular) solution contained (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 5 Mg-

ATP, pH 7.3–7.4 with KOH. From a holding potential of -80 mV, Kv7.4 currents were activated

either by 1.5 sec depolarizing pulses from -80 mV to +40 mV in +10 mV increments, followed by

an isopotential pulse at 0 mV of 500 ms duration, or by 3-sec voltage ramps from -80 mV to +20

mV applied at 0.08 Hz frequency. Current were recorded using an Axopatch-200A amplifier,

filtered at 5 kHz, and digitized using a DigiData 1440A (Molecular Devices, Union City, CA,

USA). The pCLAMP software (version 10.2) was used for data acquisition and analysis (Molecular

Devices, Union City, CA).

Electrophysiological data analysis

For Kv7.4 currents in Xenopus oocytes, whole-cell conductance (G) was calculated

according to the following equation: G=Imax/(V–EK), where I is the steady-state current measured

at the end of each depolarizing step, V is the step potential, and EK is the reversal potential for

potassium, which was calculated to be -98 mV. Conductance values were expressed as a function of

membrane potential, and fit to a single Boltzmann distribution of the following form:

y=max/[1+exp(V1/2-V)/k], where V is the test potential, V1/2 is the half-activation potential, and k is

the slope factor (Wickenden et al., 2008).

For Kv7.4 currents in CHO cells, conductance-voltage (activation) curves were generated by

normalizing to the maximal value of the instantaneous isopotential currents at 0 mV, and expressing

the normalized values as a function of the preceding voltages. Data were fit to a sum of two


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independent Boltzmann distributions (B1 and B2) of the following form: y=max1/(1+exp(V1-

V)/k1)+max2/(1+exp(V2-V)/k2), where V is the test potential, V1 and V2 the half-activation potentials,

k1 and k2 the slopes, and max1 and max2 the relative amplitudes for each Boltzmann distribution

(Castaldo et al., 2002). Ramp-evoked currents were analyzed by measuring the currents at -40 mV

(a membrane potential value close to the activation threshold) and at 0 mV (a membrane potential

value at which Kv7.4 conductance is largely saturated), and the effect of each venom fraction was

expressed as the percent of current increase at these two membrane voltages. Data analysis and fit

were performed using the Origin software (version 8.0; OriginLab, Northampton, MA, USA).

Disulfide bridges reduction and enzymatic hydrolysis

HPLC purified venom fractions were dissolved in 50 mM NH4HCO3 buffer at pH 8.0, and

dithiothreitol (DTT) was added to a final concentration of 2 mM. Samples were allowed to react at

37°C for 2 h. Enzymatic digestion was then performed by adding trypsin with an enzyme/substrate

ratio of 1/50 w/w at 37 °C for 16 h in the presence of DTT.

MALDI Mass Spectrometry

MALDI-MS experiments were performed on a Voyager-DE STR MALDITOF mass

spectrometer (Applied Biosystems, Framingham, MA, USA) equipped with a nitrogen laser (337

nm). 1 μl of the sample was mixed (1/1, v/v) with a 10 mg/ml solution of α-cyano-4-

hydroxycinnamic acid in ACN/50 mM citrate buffer, 70/30 v/v. Spectra were acquired using a mass

(m/z) range of 400-5000 amu for Peptide Mass Fingerprint and 3000-8000 amu for molecular

weight determination.

TANDEM mass spectrometry.

The peptide mixtures were filtered using 0.22 µm PVDF membrane (Millipore) and

analysed using a 6520 Accurate-Mass Q-TOF LC/MS System equipped with a 1200 HPLC system

and a chip cube (Agilent Technologies, Palo Alto, CA, USA). After sample loading, the peptide

mixture was first concentrated and washed on 40 nl enrichment column (Agilent Technologies

chip), with 0.1% formic acid in 2% ACN as the eluent. The sample was then fractionated on a C18

reverse-phase capillary column at flow rate of 400 nl/min, with a linear gradient of eluent B (0.1%

formic acid in 95% ACN) in A (0.1% formic acid in 2% ACN) from 7 to 80% in 50 min. Peptide

analysis was performed using data-dependent acquisition of one MS scan (mass range from 300 to

1800 m/z) followed by MS/MS scans of the five most abundant ions in each MS scan. Dynamic

exclusion was used to acquire a more complete survey of the peptides by automatic recognition and


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temporary exclusion (0.5 min) of ions from which definitive mass spectral data had been previously

acquired. Nitrogen at a flow rate of 3 L/min and heated to 325°C was used as the dry gas for spray

desolvation. MS/MS spectra were measured automatically when the MS signal surpassed the

threshold of 50000 counts. Double and triple charged ions were preferably isolated and fragmented

over single charged ions.

Protein identification

The acquired MS/MS spectra were transformed in mz.data format and used for proteins

identification with a licensed version of MASCOT 2.1 (Matrix Science, Boston, USA); the

calculation of FDR in this Mascot version is prone to several errors

(http://www.matrixscience.com/pdf/2011WKSHP5.pdf), thus FDR values were not calculated for

our peptides. Raw data from nanoLC–MS/MS analyses were used to query the SwissProt database

available at the Uniprot KB site (http://web.expasy.org/docs/relnotes/relstat.html); it contained a

total number of 468.851 sequences (166.149.756 residues). No taxonomic restriction was specified.

Mascot search parameters were: trypsin as enzyme, allowed number of missed cleavage 2,

oxidation of methionine (+15.99492 Da) and pyro-Glu N-term Q ( -17.02655 Da) as variable

modifications, 10 ppm MS tolerance and 0.6 Da MS/MS tolerance, peptide charge, from +2 to +3.

Spectra with a MASCOT score < 25 having low quality were rejected.

Single Ion Monitoring analysis.

Peptide mixtures, obtained as previously described, were analysed by LC-MS/MS using a

4000QTrap (Applied Biosystems, Framingham, MA, USA) coupled to a NanoLC- 2D Plus system

equipped with a cHiPLC®-Nanoflex System (Eksigent, Dublin, CA, USA). The mixture was loaded

on an Eksigent Nano cHiPLC Trap column (200µm x 0.5mm ChromXP C18-CL 3 µm 300Å) at 2

μl/min (A solvent 0.1% formic acid, loading time 10 min). Peptides were separated on an Eksigent

reverse-phase Nano cHiPLC column (75 µm x 15 cm ChromXP C18-CL 3 µm 120Å), at a flow rate

of 0.3 μl/min with a 5 to 65% linear gradient in 60 min (A solvent 0.1% formic acid, 2% ACN in

water; B solvent 0.1% formic acid, 2% water in ACN). Nanospray source was used at 2.8 kV with

liquid coupling, with a declustering potential of 80 V, using an uncoated silica tip from

NewObjectives (O.D. 150 µm, I.D. 20 µm, T.D. 10 µm). Spectra acquisition was based on a survey

Enhanced Resolution Scan at 250amu/s for the ion at m/z 722.45, 481.96, 658.40 and 439.27.

Enhanced Product Ion scans (MS/MS) were performed at 4000 amu/s and collision voltages were

calculated automatically by rolling collision energy. Data were acquired and processed using

Analyst software (Applied Biosystems, Framingham, MA, USA).


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NH2-terminal amino acid sequence determination

The NH2-terminal sequence of reduced/alkylated peptides (1 nmol) was carried out for the

first 10 cycles using an automatic liquid-phase protein sequencer (model 476A; Applied

Biosystems, Foster City, CA, USA) using Edman protein degradation, according to standard

procedures.

Sequence data analysis and statistics

Similarity searches on protein sequences were performed following BLAST analysis

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The theoretical molecular weight was calculated by

ProtParam (http://us.expasy.org/cgibin/protparam) and the trypsin potential cleavage sites were

predicted using PeptideCutter (http://web.expasy.org/peptide_cutter/).

Statistical differences between data groups, expressed as means±SEM, were tested using an

unpaired two-tailed Student’s t-test, assuming that the population follows a Gaussian distribution.

Differences were considered statistically-different vs respective controls when p<0.05.


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Results

Effects of gel filtration-purified Aa venom fractions on Kv7.4 channels expressed in Xenopus

oocytes.

To search for naturally-occurring peptides active on Kv7.4 channels from the Aa scorpion,

crude Aa venom was separated by gel filtration. The resulting profile (Fig. 1A) shows five partially-

resolved fractions; among these, four correspond to non-toxic fractions (M1, M2, M3, and M4), and

one (G50) was toxic in-vivo (Miranda et al., 1970). Fractions M1, M2 and G50, containing

molecules having a molecular weight range >2 kDa, were selected for functional screening on

Kv7.4 channels expressed in Xenopus oocytes by two microelectrode voltage-clamp; M3 and M4

fractions were not studied, as they contained low molecular weight material in relatively low

abundance. Kv7.4 currents were activated by membrane depolarization above -40 mV, and

displayed slow activation kinetics, and lack of inactivation (Fig. 1B, left panel; Fig. 1C) (Kubish et

al., 1999). Kv7.4 currents were blocked by 76±5% (n=3; +40 mV) by 100 µM of the non-selective

Kv7 channel blocker linopirdine. Oocyte perfusion for 5 minutes with M2 (50 µg/ml) caused a

38.6±2.5% increase in the Kv7.4 peak currents at +40 mV, together with a negative shift in the

midpoint potential of activation (Fig 1B, right panel; Fig. 1C). In fact, analysis of the G/V curves

for Kv7.4 currents revealed that the half activation voltage (V1/2) was -15.8±0.7 mV in controls and -

25.0±2.5 after M2 exposure (n=5; p<0.05); no significant change was instead observed in the slopes

(k) of the G/V curve between controls (14.8±0.4 mV/e-fold) and M2-treated (14.2±0.7 mV/e-fold)

G/V relationships (n=5; p>0.05). Both these effects were largely reversible after 5 min washout of

the M2 fraction. The effect of M2 on Kv7.4 channels was dose-dependent between 1 and 100 µg/ml

(Fig. 1D); higher concentrations could not be tested because of the limited material availability. In

contrast to M2, perfusion with G50 (50 µg/ml) did not modify Kv7.4 currents, whereas 50 µg/ml

M1 significantly decreased Kv7.4 current amplitude by 51.4±9% (Fig. 1E). Given our interest in

Kv7.4 activators rather than blockers, this latter effect was not pursued any further.

Effect of M2 sub-fractions on Kv7.4 channels expressed in mammalian cells.

The M2 fraction contains a heterogeneous mixture of molecules ranging from 7 to 12 kDa.

To achieve a better size-separation, the M2 fraction eluted from the Sephadex G-50 column was

collected into 4 sub-fractions (M2a, M2b, M2c, and M2d) (Fig. 2A). Given the little amount of

purified fractions available, the effect on Kv7.4 channels exerted by each M2 sub-fraction was then

studied using the whole-cell configuration of the patch-clamp technique in mammalian cells

transiently transfected with Kv7.4 cDNA; this strategy allowed to use much less material than that


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required for Xenopus oocytes experiments. In addition, to decrease the length of each experiment

and further reduce the amount of material needed, we tested the effects of the four M2 sub-fractions

using a voltage protocol in which Kv7.4 currents were activated by 3-sec voltage ramps from -80

mV to +20 mV applied at 0.08 Hz frequency. Perfusion with 50 µg/ml of the M2c sub-fraction,

corresponding to the descending flank of the M2 fraction (Fig. 2A), enhanced ramp-evoked Kv7.4

currents (Fig. 2B). As shown in Fig. 2C, the extent of Kv7.4 current enhancement by M2c was

larger at -40 mV (+44.4±3.8%) than at 0 mV (+24.5±1.7%), thus largely reproducing the

previously-described effects exerted by the whole M2 fraction on Kv7.4 channels expressed in

Xenopus oocytes. M2c was the only M2 sub-fraction active on Kv7.4 channels; in fact, perfusion

with M2a, M2b, and M2d (each at 50 µg/ml) failed to affect the threshold activation potential or the

maximal current amplitude of the expressed channels (Fig. 2C).

Effect of M2c sub-fractions on Kv7.4 channels expressed in mammalian cells.

The M2c gel filtration sub-fraction was resolved on a C8 analytical HPLC column, leading

to the identification of nine peaks (P1-P9) (Fig. 3A). When each of these purified peaks were

separately tested (each at 20 µg/ml) for their effect on Kv7.4 currents by patch-clamp experiments,

it was found that only P8, eluted at 19 min, enhanced Kv7.4 currents (Fig. 3B). Again, as already

observed for the whole M2 fraction and the M2c sub-fraction, P8 effectively increased Kv7.4

currents at every potential tested, although the enhancement was more significant around membrane

potential values closer to the activation threshold (-40 mV) than at 0 mV, a membrane potential

value at which the K+ conductance is largely saturated (Fig. 3C; see also Fig. 4B). None of the other

peak resulting from M2c purification modified Kv7.4 currents at -40 mV or 0 mV (Fig. 3B). Fig. 3C

shows a representative example of Kv7.4 current activation by cell perfusion with 20 µg/ml of P8,

using the previously-described voltage ramp protocol.

Notably, the effects of P8 on Kv7.4 currents were qualitatively similar to those of the

prototype Kv7 activator retigabine (Fig. 3B). To compare the kinetics of onset and offset of the

Kv7.4-activating effects of P8 with those of retigabine, time-course experiments were performed.

As shown in Fig. 3D, P8-induced Kv7.4 activation developed slowly, requiring about 1 minute to

reach steady-state levels; similarly, basal current values were recovered after about 1 minute upon

drug removal. By contrast, the kinetics of onset and offset of 10 µM retigabine appeared

significantly faster, requiring less than 30 seconds to reach steady-state levels (Fig. 3D). During the

4 min of 50 μg/ml P8 perfusion, the maximal response decreased by an average of 14.2±4.3%

(n=8).


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The voltage ramp protocol used in the previously-described experiments does not allow to

precisely define the effect of the P8 fraction on the steady-state properties of the Kv7.4 conductance;

therefore, in subsequent experiments, Kv7.4 channels were activated by depolarizing pulses at

constant voltages. Fig. 4A shows a family of Kv7.4 currents recorded before (left panel) and after

(right panel) P8 exposure. Activation curves revealed that P8 effectively increased both maximal

conductance and caused a leftward shift in the activation threshold of Kv7.4 currents. In particular,

experimental data analysis revealed that a sum of two Boltzmann equations (B1 and B2) was

required to properly fit the data (Castaldo et al., 2002). Under control conditions, the two

Boltzmann components had a 60/40 ration in their relative amplitude (max1 and max2 accounted for

59±4% and 42±4% of the total conductance, respectively), and distinct half activation potentials

(V1=-38.1±1.0 mV; V2=-7.3±2.1 mV) and slopes (k1= 9.2±0.6 mV/e-fold; k2= 15.0±1.3 mV/e-fold).

After P8 exposure, no significant changes were observed in the relative amplitudes (max1 and max2

accounted for 62±1% and 44±1% of the total conductance, respectively), nor in V2 (-5.1±2.3 mV) or

k2 (19.2±3.6 mV/e-fold); by contrast, both V1 (-47.7±2.4 mV) and k1 (12.2±0.5 mV/e-fold) were

significantly different from controls (n=5; p<0.05). On the other hand, retigabine failed to affect the

relative amplitudes (max1 and max2 accounted for 66±12% and 35±12% of the total conductance,

respectively), or the slopes (k1=11.1±1.8 mV/e-fold ; k2=11.1±2.2 mV/e-fold)) of B1 and B2; the

midpoint potentials of both Boltzmann distribution were shifted in the hyperpolarizing direction by

retigabine (V1=-57.5±2.1 mV; V2=-29.4±6.8 mV) (n=5 ; p<0.05). The inset of Fig. 4B compares the

normalized activation curves for control, P8 and retigabine.

Finally, within the tested range (5-50 μg/ml), the effect of P8 on Kv7.4 current enhancement

at -40 mV was concentration-dependent (Fig. 4C).

Mass spectral analysis of the P7 and P8 fractions.

Given that P8 contained the biologically-active molecule(s) of interest, this fraction was

directly submitted to MALDI-TOF mass spectrometry analysis; the same analysis was also

performed on P7, which despite showing an HPLC elution profile very close to P8, was unable to

modify Kv7.4 currents. MALDI-MS spectra revealed the occurrence of a major signal having a m/z

value of 6991.42 (Fig. 5A) and 7119.15 (Fig. 5B) in P8 and P7, respectively. Both P8 and P7

fractions were subjected to disulfide bridges reduction and trypsin digestion, and the obtained

peptide mixture analysed by MALDI-MS and LC-MS/MS. LC-MS/MS data analysis led to the

identification in both fractions of AaTXKβ from Androctonus australis (UniProtKB P69939),

whose sequence was inferred from an Aa cDNA library (Legros et al., 1998). Figures 5C and 5D

report the P8 and P7 fractions sequence coverage obtained by both MALDI-MS and LC-MS/MS


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experiments, mapped onto the AaTXKβ mature sequence. In both P8 and P7 fractions, the N-

terminus peptide escaped mass spectral analysis. Since the mass difference detected in MALDI

analysis between P8 and P7 major species (127.73 Da) could well be due to the removal during the

pro-protein maturation of the N-terminal lysine residue (expected molecular mass 128.18 Da), P8

and P7 mixtures were submitted to LC-MS/MS analysis in Single Ion Monitoring (SIM) mode (Fig.

6), searching for both peptides 1-13 (KLVKYAVPVGTLR) and 2-13 (LVKYAVPVGTLR). In P7,

the presence of the signal related to peptide 1-13 and the contemporary absence of the signal related

to the peptide 2-13 (Figs. 6B and 6D) demonstrated the occurrence of the full-length AaTXKβ in

this fraction. On the other hand, SIM analysis revealed the presence of the signal relative to peptide

2-13 as the major specie in P8 (Figs. 6A and 6C). A good sequence coverage for both 2-13 and 1-13

peptides was obtained by the occurrence of intense y-ion series signals as shown in Figs. 6C and

6D. However, no signals attributable to fragment peaks that appear to extend from the amino

terminus (“b ions”) for both P7 and P8 peptides could be detected in the mass tandem spectra,

probably due to the physicochemical characteristics of the peptides.

Since MS/MS data did not unambiguously identify the N-terminus sequences, Edman

degradation of S-pyridyl-ethylated P8 and P7 was performed, leading to the identification of the N-

terminal amino acid partial sequences LVKYAV and KLVKYAV in P8 and P7, respectively.

Altogether, results from molecular weight determination, mass mapping analysis, SIM analysis, and

Edman degradation strongly suggested that the P8 peptide active on Kv7.4 channels corresponds to

a novel variant of AaTXKβ which lacks the N-terminal lysine; this peptide is thus called

AaTXKβ(2-64). By contrast, the same experimental procedures revealed that the inactive P7 fraction

corresponded to the full-length sequence of AaTXKβ(1−64).

Only a monomeric form was observed in the mass spectra of both AaTXKβ(1−64) and

AaTXKβ(2-64) polypeptides, suggesting that no intermolecular disulphide bridge were formed. These

results indicate that the six cysteins of AaTXKβ(1-64) and AaTXKβ(2-64) are engaged in three

intramolecular disulphide bridges, as previously hypothesized (Legros et al., 1998).

Effect of P8 on Kv7.1, Kv7.2, Kv7.3, Kv7.2/3, Kv7.5/3, and Kv1.1 currents

To investigate whether, in addition to Kv7.4 channels, other K+ channels were also sensitive

to AaTXKβ(2−64), experiments were performed in CHO cells expressing homomeric Kv7.1, Kv7.2,

Kv7.3, and Kv1.1 channels, as well as heteromeric Kv7.2/3 and Kv7.5/3 channels. Given that the

results obtained by MALDI-TOF, LC-MS/MS, SIM and EDMAN experiments revealed that

AaTXKβ(2−64) was the major component of P8, in consideration of the little amount of P8 available


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for the experiments, the P8 fraction was used for these experiments, thus avoiding another step of

AaTXKβ(2−64) purification.

When activated by the described voltage ramp protocol, Kv7.3 and Kv7.2/3 channels showed

similar sensitivity as Kv7.4 to modulation by P8 (Fig. 7A). In fact, at potentials close to threshold

values (-40 mV), P8 significantly increased Kv7.3, Kv7.2/3, and Kv7.4 currents by about 70% (Fig.

7B). Noteworthy, at more depolarized potentials (0 mV), P8 only increased the maximal currents in

homomeric Kv7.4 and heteromeric Kv7.2/3 channels, whereas no effect was observed on

homomeric Kv7.3 channels (Figs. 7A and 7B). Moreover, 20 μg/ml P8 enhanced the currents

carried by Kv7.5/3 heteromeric channels at 0 mV by 25.5±1.4 % (n=4). Currents at -40 mV in

Kv7.5/3-transfected cells, as well as currents at any potential in cells transfected only with Kv7.5

cDNA from three different sources (see Materials and Methods), were too small to assess the effects

of P8 (data not shown). Finally, P8 failed to modify current amplitudes at depolarized potentials or

activation threshold in Kv7.1, Kv7.2 and Kv1.1 channels (Figs 7A and 7B).


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Discussion

In the present work we found that the non-toxic fraction from the venom of the North

African scorpion Androctonus australis (Aa) (called M2) dose-dependently enhanced Kv7.4

currents in Xenopus oocytes, and caused a 10 mV negative shift in the midpoint potential of the

activation curve. Biological activities of peptides from non-toxic venom fractions have been

previously described in Buthus occitanus tunetanus scorpion venom; these include lipolytic

properties (Soudani et al., 2005), activation of nicotinic receptors in skeletal muscle cells (Cheikh et

al., 2007), and inhibition of cardiac L-type calcium currents (Cheikh et al., 2006).

Within the 9 HPLC fractions of M2, only the sub-fraction called P8 displayed Kv7.4-

activating properties. Indeed, P8 caused an increase in the maximal currents, and hyperpolarized

and decreased the steepness of the activation curve of Kv7.4 channels. In P8, mass mapping data

identified AaTXKβ, a long chain β-KTX whose sequence was inferred upon screening of a cDNA

library from Aa venom glands (Legros et al., 1998). Surprisingly, same analysis performed on the

P7 HPLC peak, which did not enhance Kv7.4 currents, also led to the identification of

AaTXKβ. The data obtained show that two different forms of AaTXKβ, having very distinct

biological activities and differing by 127.73 Da in molecular mass, were present in P7 and P8

fractions. The combined results from SIM analysis and Edman degradation experiments revealed

that the most abundant peptide in the P7 fraction unable to activate Kv7.4 currents was full-length

AaTXKβ(1-64), whereas the P8 AaTXKβ component active on Kv7.4 currents lacked the first N-

terminal lysine (AaTXKβ(2-64)). Thus, the removal of the N-terminal residue from the protein mature

form, possibly as a further step in the maturation process, does infer this dramatic functional

change.

The biological functions of β-KTx’s are poorly characterized (Diego-Garcia et al., 2007).

Selective interference with Kv channels has been verified only for TsTXKβ (inhibition of 86Rb+

efflux carried by non-inactivating K+ channels in rat brain synaptosomes; Rogowski et al., 1994),

BmTXKβ (blockade of ITO current in rabbit atrial myocytes; Cao et al., 2003a), MeuTXKβ1

(inhibition of 125I-kaliotoxin binding to rat brain synaptosomal membranes; Zhu et al., 2010), and a

chemically-digested C-terminal part of HgeβKTX (reduction of Kv1 channel currents expressed in

Xenopus oocytes; Diego-Garcia et al., 2008). By contrast, no study has been carried out to isolate

the corresponding peptide from Aa venom, and the activity of AaTXKβ on K+ channels (or on any

ion channel) has never been investigated. AaTXKβ has a high sequence similarity (~88% identity)

with MeuTXKβ1 from Mesobuthus eupeus (Fig.8), a peptide showing weak affinity for K+

channels, but exhibiting anti-Plasmodial and cytolytic activities, and whose structural features have


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been resolved (Zhu et al., 2010). It has been suggested that, when compared to TsTXKβ (Rogowski

et al., 1994), the weak affinity of MeuTXKβ1 for K+ channels may be explained by its extended N-

terminal domain with 9 extra residues, highlighting a critical role of this region for target

recognition. AaTXKβ also lacks this N-terminal extension, and, similarly to TsTXKβ, could be a

‘truncated’ version of MeuTXKβ1. Notably, a critical influence of the N-terminal region for

scorpion toxins interaction with K+ channels has been already described for KTX2 (Legros et al.,

1997), and for HgeβKTX (Diego-Garcia et al., 2008). Similarly, chemical modifications and

mutagenesis studies have revealed that the N-terminal amino group or the N-terminal basic residues

of scorpion β-neurotoxins (or both) are critical for their interaction with voltage-gated Na+ channels

(Polikarpov e al., 1999).

Altogether, these data suggest that β-KTx’s having the classical Cs-α/β scaffold not only

produce inhibitory effects on K+ channels, but can also activate ionic conductances. This ability has

been previously described for martentoxin (MarTx), an α-KTx purified from the East-Asian

scorpion Mesobuthus martensi venom, which enhances the activities of native and cloned BK

channels (Tao et al., 2011), and DkTx (double-knot toxin) purified from the venom of the Chinese

bird tarantula Ornithoctonus huwena, which selectively and irreversibly activates transient receptor

potential vanilloid-1 (TRPV1) channels (Bohlen et al., 2010). Notably, a MarTx variant (MarTx-1)

lacking of the N-terminal phenylalanine displayed similar biological activities when compared to

the full-length MarTx (Cao et al., 2003b).

The ability of AaTXKβ(2-64) to cause an hyperpolarization shift in Kv7.4 channels gating is

reminiscent of the effects exerted on voltage-gated Na+ channels by β-scorpion toxins acting at site

4 (Dutertre and Lewis, 2010); these toxins enhance voltage-dependent activation of Na+ channels by

modifying the movement of the S4 segments via a voltage-sensor trapping mechanism (Catterall et

al., 2007). Toxin-induced sensor-trapping can also occur in voltage-gated Ca2+ channels (agatoxins

from spiders) (McDonough, 2007), and in voltage-gated K+ channels of the Kv2 and Kv4

(hanatoxins from tarantula) (Swartz and MacKinnon, 1997), or Kv3 and Kv11 (sea anemone toxins)

(Diochot and Lazdunski, 2009) subclasses. It has been reported that a conserved structural motif in

voltage-gated K+ and Ca2+ channels can account for the “promiscuity” of both hanatoxin and

grammotoxin (another tarantula toxin) to interact with both channel classes (Li-Smerin and Swartz,

1998), and, possibly, with non-voltage gated channels of the TRPV1 sub-family (Siemens et al.,

2006).

Distinct similarities and difference can be found between AaTXKβ(2-64) and previously

described synthetic Kv7 channels openers. In fact, similarly to retigabine, the prototype Kv7 opener

in clinical use as an anticonvulsant (Miceli et al., 2008), AaTXKβ(2-64) hyperpolarizes the activation


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threshold voltage and increases the maximal currents of Kv7.4 channels; however, at the

concentrations tested, the efficacy of AaTXKβ(2-64) on Kv7.4 currents seems lower than that of

retigabine. The potency of retigabine in activating channels formed by distinct Kv7 subunits appears

slightly different, with Kv7.2 and Kv7.3 showing higher (EC50<1μM), and Kv7.4 and Kv7.5 lower

(EC50>1μM) drug sensitivity (Tatulian et al., 2001; Dupuis et al., 2002). However, since these

differences are quantitatively rather small, retigabine is currently regarded as a poorly selective

opener for channels composed of Kv7.2-5 subunits, being instead ineffective in Kv7.1 channels. In

the present experiments, in addition to homomeric Kv7.4, AaTXKβ(2-64) produced a hyperpolarizing

shift in opening voltage-dependence through homomeric Kv7.3 and heteromeric Kv7.2/3 channels.

At potential above 0 mV, AaTXKβ(2-64) increased the maximal current in homomeric Kv7.4 and

heteromeric Kv7.2/3 and Kv7.5/3 channels, but not in homomeric Kv7.3 channels. Strikingly, both

homomeric Kv7.1 and Kv7.2, as well as the distantly-related Kv1.1 channels were unaffected by

AaTXKβ(2-64). In Kv7.4 channels, the effect of AaTXKβ(2-64) was relatively slow compared to

retigabine and largely reversible, possibly suggesting an extracellular site of action.

Multiple mechanisms might cause the subtype-selective macroscopic current increase at

depolarized voltages by AaTXKβ(2-64) in Kv7 channels; these include an increase in the number of

functional channels, of the single channel conductance, or of the maximal channel open probability

(Po). The possibility that AaTXKβ(2-64) recruits more active channels at the plasma membrane seems

unlikely, given that the toxin actions were rather fast, requiring about one minute to achieve

maximal effect, and were largely reversible. Considering that most toxins acting as gating

modulators of Kv channels bind at a distance from the pore and do not affect single-channel

conductance (Swartz and MacKinnon, 1997), we therefore speculate that AaTXKβ(2-64) increases

the Po of Kv7.4 channels. Although single channel experiments were not performed to solve this

issue, the fact that AaTXKβ(2-64) caused an hyperpolarization shift with no significant augmentation

of maximal currents in Kv7.3 channels whose maximal Po is close to unity (Li et al., 2004) seems in

line with such speculation.

Recently, acidic scorpion toxins belonging to new κ-KTX2 and α-KTX28 subfamilies have

been found to inhibit Kv7.1 channels, although they did not affect other K+ channels which are

canonical targets for basic KTx (such as Kv1.3); unfortunately, the pharmacological profile of these

toxins for channels formed by other Kv7 subunit combination is currently unknown (Chen et al.,

2012). Other compounds with a certain degree of selectivity for Kv7 subtypes are: 1. the acrylamide

derivative (S)-1 which preferentially activates Kv7.4 and Kv7.5 channels (Bentzen et al., 2006); 2.

the substituted benzamide ICA-27243, which selectively increased the maximal conductance and

negatively-shifted the steady-state activation curve in Kv7.2/Kv7.3 heterotetramers, with lower


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potency on Kv7.4 or Kv7.3/Kv7.5 channels and no activity on Kv7.1 channels (Wickenden et al.,

2008); 3. the diphenylamine carboxylate derivative NH29, which increases the current amplitude of

Kv7.2, is weakly effective in Kv7.4, and is ineffective on Kv7.3 and Kv7.1 channels (Peretz et al.,

2010); 4. the cysteine-alkylating agent N-ethylmaleimide, which increases the currents carried by

Kv7.2, Kv7.4 or Kv7.5, but not by Kv7.1 and Kv7.3 channels (Li et al., 2004); 5. zinc pyrithione,

which potently activates Kv7.1, Kv7.2, Kv7.4, and Kv7.5, but not Kv7.3, channels (Xiong et al.,

2007). Notably, the existence of at least two different binding regions, one in the pore and/or in the

gating hinge and one in the voltage sensor itself, appears to contribute to the pharmacological

profile shown by synthetic openers for distinct Kv7 subunit combinations (Miceli et al., 2011);

whether these sites also contribute to the selectivity pattern of AaTXKβ(2-64) remains to be

investigated.

In conclusion, in the present study, we report the isolation and functional characterization of

AaTXKβ(2-64), a novel variant of AaTXKβ from the non-toxic fraction of the Androctonus australis

scorpion acting as the first peptide activator of Kv7 channels. We anticipate that these results might

prove useful to unravel novel biological roles of AaTXKβ(2-64)-sensitive Kv7.4 channels, and to

develop novel pharmacological tools allowing subtype-selective targeting of Kv7 channels.


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Acknowledgements

We are indebted to Prof. Thomas J. Jentsch (Max-Delbrück-Center for Molecular Medicine,

Berlin, Germany) for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 cDNAs in pcDNA3.1, Prof. Mark Shapiro

(University of Texas Health Science Center, San Antonio, TX, USA) for Kv7.5 cDNA in

pcDNA3zeo, Prof. Anastasios Tzingounis (University of Connecticut, Storrs, CT, USA) for Kv7.5

cDNA in a pIRES2-DsRed-Express vector, Prof. Enzo Wanke (University of Milan Bicocca, Milan,

Italy) for Kv1.1 cDNA, and Dr. Eckhard Ficker (Rammelkamp Center, Cleveland, OH, USA) for

Kv7.1 cDNA. We also thank Professor Hechmi Louzir (General Director of the Pasteur Institute of

Tunis) and Dr Najet Srairi-Abid (Venoms and therapeutic molecules Laboratory, Pasteur Institute

of Tunis) for help during the biochemical purification of P8, Dr. Zakaria Ben Lasfar and his

collaborators (Veterinary Laboratory, Pasteur Institute of Tunis) for providing Aa venom and

Xenopus care, and Dr Mejdoub Hafedh (University of Sciences, Sfax) for Edman degradation

experiments. We are also grateful to Dr. Kathy-Ann Koralek and to the Dargut and Milena Kemali

Foundation for organizing and sponsoring, respectively, the First Kemali-IBRO Mediterranean

School of Neuroscience held in Naples, on September 21-30, 2009, where this project was

conceived, and to Prof. Lucio Annunziato (Dept. of Neuroscience, University of Naples Federico II,

Naples, Italy) for his generous support to the project.

Author Contributions:

Participated in research design: Marino, El Ayeb, Taglialatela, Benkhalifa

Conducted experiments: Landoulsi, Miceli, Palmese, Amoresano

Performed data analysis: Landoulsi, Miceli, Palmese, Amoresano, Marino, Taglialatela, Benkhalifa

Contributed to the writing of the manuscript: Landoulsi, Amoresano, Marino, Taglialatela,

Benkhalifa


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two-domain potassium channel toxin-like peptide with cytolytic activity. Biochim Biophys Acta 1804:872-883.


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Footnote

The present study was supported by grants from the Pasteur Institute of Tunis, Ministry of Health

and Research & Development [Tox11] (to RB); Telethon [GGP07125], the Fondazione San Paolo–

IMI [Project Neuroscience], Regione Molise [Convenzione AIFA/Regione Molise], Italian Ministry

for Education and Research [PRIN 2009] (to MT); and Regione Campania 2013 [” PON01_01802”

and PON01_00117] (to AA). ZL received a traineeship grant from the International Division of the

Pasteur Institute [RIIP/EC/MAM/N° 117/11]. The authors declare no competing financial interests.


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Figure Legends

Fig. 1. Effects of Aa venom purified gel-filtration fractions on Kv7.4 channels expressed in Xenopus

oocytes. (A) Gel filtration chromatogram of the Aa venom. (B) Effect of the M2 fraction on Kv7.4

currents in a representative cells. Currents traces from Kv7.4 channels were recorded before (left

panel) and 5 min after the application of M2 (50 µg/ml; right panel). As shown in the panel,

currents were activated by 2 s depolarizing pulses from a holding potential of -80 mV to potentials

ranging from -100 mV to +40 mV in 10 mV increments, followed by 300 ms step to -120 mV.

Scale bar: vertical 0.5 μA, horizontal 0.5 sec. (C) Effect of the M2 fraction (50 µg/ml) on the

activation curve of Kv7.4 channels (n= 5). The solid lines represent the fits of the experimental data

to a Boltzmann distribution (see Methods). The dotted line is the M2 data fit trace normalized to

controls. (D) Concentration-dependent effects of M2 on Kv7.4 currents. The calculated EC50 value

is 58 µg/ml and the Hill coefficient is 1.6. (E) Effect of M1, M2 and G50 Aa fractions (each at 50

µg/ml) on Kv7.4 currents at +10 mV (n=5 for M1; n=8 for M2; and n=4 for G50; *p<0.05 vs

controls).

Fig. 2. Effect of M2 sub-fractions on Kv7.4 channels expressed in mammalian cells. (A) The gel

filtration chromatogram of fraction M2 was divided into four sub-fractions (M2a, M2b, M2c, and

M2d). (B) Kv7.4 currents expressed in CHO cells were activated by a 3 s voltage ramp from -80 mV

to +20. Recording were obtained before and after the application of M2c (50 µg/ml). The dotted

line indicates the zero-current level. Scale bar: vertical 0.2 nΑ, horizontal 500 msec. (C) Effect of

the 4 M2 sub-fractions on Kv7.4 currents at -40 mV (filled bars) or 0 mV (empty bars; n=3 for M2a

and M2d; n=5 for M2b; and n=6 for M2c; *p<0.05 relative to controls).

Fig. 3. Effect of M2c HPLC sub-fractions on Kv7.4 channels expressed in mammalian cells. (A)

HPLC separation of M2c. 50 µg of M2c was loaded per HPLC run on an analytical C8 reverse-

phase column and separated using a gradient of buffer B (0.1% trifluoroacetic acid in acetonitrile)

as described in experimental procedures and represented in the figure by a dotted line. Collected

fractions were labeled with numbers (from 1 to 9). (B) Effect of the nine HPLC peaks (each at 20

µg/ml) purified from M2c and of retigabine (10 µM, RTG) on Kv7.4 current amplitude at -40 mV

(filled bars) or 0 mV (empty bars) relative to controls (n=3 for P1, P6, P9; n=4 for P3; n=5 for P2,

P4, P5; n=6 for P7; n=19 for P8; and n=10 for RTG). Currents were expressed as percent of the

control current amplitude at -40 mV and 0 mV for each fraction; statistical comparison for each

fraction was performed relative to their respective controls (*p<0.05). (C) Ramp-evoked Kv7.4


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currents recorded before and after the application of 20 µg/ml of P8. The dotted line indicates the

zero-current level. Scale bar: vertical 0.2 nΑ, horizontal 500 msec. (D) Time course of Kv7.4

current potentiation by P8 (50 µg/ml) and retigabine (10 µM, RTG). The steady-state amplitudes of

the peak currents at -40 mV were plotted as a function of time. The bars on top of the plot indicate

the duration of drug application.

Fig. 4. Effect of P8 on the activation curve of Kv7.4 channels expressed in CHO cells, and dose-

dependent enhancement of the currents at -40 mV by P8. (A) Representative macroscopic current

traces recorded from a CHO cell expressing Kv7.4 homomeric channels in response to the voltage

protocol indicated, before (left) and after (right) application of 20 μg/ml P8. Scale bar: vertical 0.5

nΑ, horizontal 500 msec. (B) Steady-state activation curves for Kv7.4 currents before (empty

symbols) and after (filled symbols) application of 20 μg/ml P8 (n=6). Continuous lines represent

fits of the experimental data to a double Boltzmann distribution. The inset shows a comparison

among the normalized activation curves for P8 (20 μg/ml; dashed line) and RTG (10 µM; dotted

line) and those of controls (continuous line). (C) Concentration-dependent effects of P8 on Kv7.4

currents calculated at -40 mV (n=3 for 5 μg/ml; n=4 for 10 μg/ml; n=19 for 20 μg/ml; and n=8 for

50 μg/ml). Asterisks indicate values significantly different (p<0.05) vs controls.

Fig. 5. Biochemical characterization of C8 HPLC-eluted peptides in P8 and P7. (A, B) Molecular

weights of purified peptides determined by MALDI-TOF in P8 (A), and P7 (B). (C, D) Amino acid

sequences of P8 (C) and P7 (D) determined by LC-MS/MS are in bold and underlined, and mapped

onto the full-length sequence of AaTXKβ(1-64).

Fig. 6. LC-MS/MS analysis in Single Ion Monitoring mode of the N-terminal region of HPLC-eluted

peptides in P8 and P7. (A-B) Total ion chromatogram of MS/MS analysis in Single Ion Monitoring

mode of P8 (A) and P7 (B) searching for peptides 2-13 (LVKYAVPVGTLR) and 1-13

(KLVKYAVPVGTLR,) in both samples. Spectra acquisition was based on a survey Enhanced

Resolution Scan at 250amu/s for the ion at m/z 722.45, 481.96, 658.40 and 439.27. (C-D) The

MS/MS spectra corresponding to the major species detected in SIM analyses are reported and

properly annotated. Enhanced Product Ion scans (MS/MS) were performed at 4000 amu/s.

Fig. 7. Differential effect of P8 on currents carried by distinct potassium channels. (A) Representative

whole-cell current traces from Kv7.1, Kv7.2, Kv7.3, Kv7.2/3, Kv7.4 and Kv1.1 channels activated by

the previously described ramp protocol (except for Kv1.1 in which the duration of the ramp protocol


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from -80 to +20 mV was 250 ms instead of 3 s). Asterisks indicate currents recorded after perfusion

with P8 (20 µg/ml). In each panel, the dotted lines indicate the zero-current level. (B) Effect of P8

(20 µg/ml) on current amplitudes of the indicated channels at -40 mV (filled bars) or 0 mV (empty

bars) (n=3 for Kv7.1; n=6 for Kv7.2; n=3 for Kv7.3; n=5 for Kv7.2/3; n=19 for Kv7.4; and n=3 for

Kv1.1; *=p<0.05).

Fig. 8. Amino acid sequence of the major components of P8 and P7, and comparison with other β-

KTx’s. Amino acid sequence determined from MALDI-MS + LC/MS-MS, LC-SIM, and Edman

degradation results of native P8 (AaTXKβ(2-64)) and P7 (AaTXKβ(12-64); Legros et al., 1998) were

aligned with analogous toxins: BmTXK-β-2 (Zhu et al., 1999) from Mesobuthus martensii,

BuTXK-β from Buthus occitanus israelis, MeuTXK-β1 and MeuTXK-β2 (Zhu et al., 2010) from

Mesobuthus eupeus, Tdi-β-KTX and TdiKIK (Diego-Garcia et al., 2007) from Tityus discrepans,

Tco-β-KTX and TcoKIK (Diego-Garcia et al., 2005) from Tityus costatus, Ttr-β-KTX and TtrKIK

from Tityus trivittatus (Diego-Garcia et al., 2007), Hge-scorpine from Hadrurus gertschi (Diego-

Garcia et al., 2008), TsTXK-β from Tityus serrulatus (Rogowski et al., 1994). Gaps (–) have been

introduced to optimize alignment. Signal peptide and pro-peptide sequences are not reported.

Residues are colored according to the following scheme: magenta, basic; blue, acid; red, non polar;

green, polar. The six highly-conserved C residues are highlighted in green. (|) indicates trypsin

cleavage sites after marked amino acids for both native P8 (AaTXKβ(2-64)) and P7 (AaTXKβ(1-64)).


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a novel toxin variant from the Androctonus australis scorpion...

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