In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 IC50 of 8 p
Trang 1Synthesis and characterization of Pi4, a scorpion toxin
Sarrah M’Barek1, Amor Mosbah1, Guillaume Sandoz2, Ziad Fajloun1, Timoteo Olamendi-Portugal3,
Herve´ Rochat1, Franc¸ois Sampieri1, J In˜aki Guijarro4, Pascal Mansuelle1, Muriel Delepierre4,
Michel De Waard2and Jean-Marc Sabatier1
1
Laboratoire International Associe´ d’Inge´nierie Biomole´culaire et Laboratoire de Biochimie CNRS UMR 6560,IFR Jean Roche, Faculte´ de Me´decine Nord,Marseille,France;2Laboratoire Canaux Ioniques et Signalization,Equipe mixte INSERM 99–31, CEA,Grenoble,France;3Institute of Biotechnology,National Autonomous University of Mexico,Cuernavaca,Mexico;
4
Unite´ de RMN des Biomole´cules,De´pt de Biochimie Structurale et Chimie,Institut Pasteur,CNRS URA 2185,Paris,France
Pi4 is a 38-residue toxin cross-linked by four disulfide bridges
that has been isolated from the venom of the Chactidae
scorpion Pandinus imperator Together with maurotoxin,
Pi1, Pi7 and HsTx1, Pi4 belongs to the a KTX6 subfamily of
short four-disulfide-bridged scorpion toxins acting on K+
channels Due to its very low abundance in venom, Pi4 was
chemically synthesized in order to better characterize its
pharmacology and structural properties An enzyme-based
cleavage of synthetic Pi4 (sPi4) indicated half-cystine
pair-ings between Cys6–Cys27, Cys12–32, Cys16–34 and Cys22–
37, which denotes a conventional pattern of scorpion toxin
reticulation (Pi1/HsTx1 type) In vivo, sPi4 was lethal after
intracerebroventricular injection to mice (LD50 of 0.2 lg
per mouse) In vitro, addition of sPi4 onto Xenopus laevis
oocytes heterologously expressing various voltage-gated K+
channel subtypes showed potent inhibition of currents from
rat Kv1.2 (IC50 of 8 pM) and Shaker B (IC50 of 3 nM) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels The sPi4 was also found to compete with
125I-labeled apamin for binding to small-conductance Ca2+ -activated K+(SK) channels from rat brain synaptosomes (IC50value of 0.5 lM) sPi4 is a high affinity blocker of the Kv1.2 channel The toxin was docked (BIGGERprogram) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the inter-action and the associated blockage of the Kv1.2 channel Keywords: Pi4; scorpion toxin; K+channels; half-cystine pairings; molecular docking
Pi4 is a K+channel-acting toxin that was isolated from the
venom of scorpion Pandinus imperator [1] It is a 38-mer
peptide cross-linked by four disulfide bridges, and therefore
belongs to the a KTX6 subfamily [2] of short-chain
four-disulfide-bridged scorpion toxins acting on K+channels
The highest sequence identities of Pi4 are shared with
members of this structural subfamily, i.e 68% with
Mauro-toxin (Scorpio maurus palmatus) [3,4], 66% with Pi7
(P imperator) [1], 61% with Pi1 (P imperator) [5–7] and
45% with HsTx1 (Heterometrus spinnifer) [8,9] The
pri-mary structure of Pi4 also contains a variant of the
consensus sequence of scorpion toxins (i.e […]C1[…]
C2XXPC3[…]C4[…](A/S/G)XC5[…]C6XC7[…]C8 instead
of […]C1[…]C2XXXC3[…](G/A/S)XC4[…]C5XC6[…] for three-disulfide-bridged toxins) that is representative of toxins from the a KTX6 structural subclass Both the classical and variant consensus sequences are thought to be responsible for folding of toxins according to a common a/b scaffold [10–13] independent of the toxin size and pharma-cology (except for Ca2+channel-acting toxins which fold according to an inhibitor cystine knot motif [14,15]) Recent1H-NMR analysis showed that synthetic Pi4 (sPi4) indeed exhibits the a/b scaffold [16] This motif, from which arises the great functional diversity of scorpion toxins, is mainly composed of an a-helix connected to a b-sheet structure (two or three strands) by two disulfide bridges The first report on native Pi4 by Olamendi-Portugal et al [1] provided some preliminary data on its pharmacology: (a) it blocked completely and reversibly voltage-gated ShakerB channel expressed in Sf9 insect cells, at 100 nM
toxin concentration (IC50 value of 8 nM), and (b) it competed with 125I-labeled noxiustoxin (Centruroides noxius) for binding on rat brain synaptosomal membranes, with an IC50value of approximately 10 nM
Here, we report the first chemical synthesis of Pi4 in order
to better characterize its pharmacology on the various K+ channel subtypes generally recognized by toxins of the
a KTX6 subfamily, i.e insect Shaker B, rat SK, Kv1.1, Kv1.2 and Kv1.3 We also investigated the disulfide bridge
Correspondence to J.-M Sabatier, Laboratoire de Biochimie CNRS
UMR 6560, et Laboratoire International Associe´ d’Inge´nierie
Biomole´culaire, IFR Jean Roche, Faculte´ de Me´decine Nord,
Bd Pierre Dramard, 13916 Marseille Cedex 20, France.
Fax: +33 491 657595, Tel.: + 33 491 698852,
E-mail: sabatier.jm@jean-roche.University-mrs.fr
Abbreviations: HsTx1, toxin 1 from the scorpion Heterometrus
spin-nifer; Kv channel, mammalian voltage-gated K + channel; Pi1, Pi4,
Pi7, toxin 1, 4 or 7 from the scorpion Pandinus imperator; SK channel,
small-conductance Ca 2+ -activated K + channel; sPi4, synthetic Pi4.
(Received 7 April 2003, revised 11 June 2003, accepted 8 July 2003)
Trang 2organization of sPi4 as two distinct patterns of reticulation
have been described for these toxins hitherto, which are
herein referred to as the maurotoxin (C1–C5, C2–C6, C3–C4
and C7–C8) [3] and Pi1/HsTx1 (C1–C5, C2–C6, C3–C7and
C4–C8) [7,9] types Because sPi4 was found to be active at
a picomolar concentration range on rat Kv1.2 channel, we
detailed the interaction of the toxin with the latter channel
by in silico docking experiments The 1H-NMR solution
structure of sPi4 [16] and a molecular model of the rat Kv1.2
channel pore region (S5–H5–S6 portion) were used for this
purpose The data were used to generate a functional map
of Pi4 towards this channel, highlighting some key residues
of this interaction such as those belonging to the toxin
functional dyad (a well-defined pair of residues that is
thought to be crucial for toxin blocking efficacy towards
the voltage-gated K+channels)
Experimental procedures
Materials
N-a-Fmoc-L-amino acids, 4-hydroxymethylphenyloxy
(HMP) resin, and reagents used for solid-phase chemical
synthesis of Pi4 were purchased from Perkin-Elmer
Organic solvents were analytical grade products from
SDS Enzymes (trypsin, and chymotrypsin) were obtained
from Boehringer Mannheim
Solid-phase synthesis of sPi4
The sPi4 was synthesized by the solid-phase method [17]
using an automated peptide synthesizer (model 433A,
Applied Biosystems Inc.) Peptide chains were assembled
by conventional stepwise synthesis on 0.3 molar equivalents
of HMP resin (1% cross-linked; 0.69 molar equivalents of
amino group g) using 1 mmol of
N-a-fluorenylmethyloxy-carbonyl (Fmoc) amino acid derivatives [7,18] The
side-chain protecting groups of sPi4 trifunctional residues were:
tert-butyl (t-Bu) for Ser, Thr, Tyr, Asp, and Glu; trityl (Trt)
for Cys, Asn, and Gln; pentamethylchroman (Pmc) for
Arg, and tert-butyloxycarbonyl (Boc) for Lys N-a-Amino
groups were deprotected by treatments with 18% and 20%
(v/v) piperidine/N-methylpyrrolidone for 3 and 8 min,
respectively The peptide resin was washed with
N-methyl-pyrrolidone (5· 1 min), then Fmoc-amino acid derivatives
were coupled (20 min) as their hydroxybenzotriazole active
esters (OBt) in N-methylpyrrolidone (3.3-fold excess) After
complete peptide chain assembly and removal of the
N-terminal Fmoc group, the peptide-resin (c 2.1 g) was
treated under stirring for 3 h at room temperature with a
mixture of 88% trifluoroacetic acid/5% H2O/5%
thioani-sole/2% ethanedithiol (v/v) in the presence of crystalline
phenol (2.5 g) in a final volume of 30 mLÆg)1 of peptide
resin The peptide mixture was filtered to remove the resin,
and the filtrate was precipitated and washed twice with cold
diethyl ether After centrifugation at 2800 g for 12 min, the
supernatant was discarded and the crude peptide was
dissolved in H2O and lyophilized Oxidative folding of the
reduced peptide was performed by dissolving the lyophilized
peptide ( 1 mM final concentration) in 0.2M Tris/HCl
buffer, pH 8.4 and gentle stirring under air for 72 h at 25C
The sPi4 was purified to homogeneity by semipreparative
reverse-phase HPLC (Perkin-Elmer, C18 Aquapore ODS
20 lm, 250· 10 mm) by means of a 60 min linear gradient
of 0.08% trifluoroacetic acid/0% to 35% acetonitrile (v/v)
in 0.1% trifluoroacetic acid/H2O (v/v) at a flow rate of
6 mLÆmin)1 (k¼ 230 nm) The correct identity and the high degree of homogeneity of sPi4 were established by: (a) analytical C18reverse-phase HPLC (Chromolith RP18,
5 lm, 4.6· 100 mm) using a 40-min linear gradient of 0.08% trifluoroacetic acid/0–60% acetonitrile (v/v) in 0.1% (v/v) trifluoroacetic acid/H2O (v/v) at a flow rate
of 1 mLÆmin)1; (b) amino acid composition after acidolysis [6MHCl/2% phenol (w/v), 20 h, 118C, N2atmosphere); (c) Edman sequencing; and (d) molecular mass analysis by MALDI-TOF mass spectrometry
Assignment of sPi4 half-cystine pairings The sPi4 (800 lg) was incubated with a mixture of trypsin and chymotrypsin at 10% (w/w) each, in 0.2M Tris/HCl buffer, pH 7.4 (14 h, 37C) The resulting peptides were separated by analytical reverse-phase HPLC (Chromolith RP18, 5 lm, 4.6· 100 mm) with a 60-min linear gradient
of 0.08% (v/v) trifluoroacetic acid/0–40% acetonitrile in 0.1% (v/v) trifluoroacetic acid/H2O at a flow rate of
1 mLÆmin)1(k¼ 230 nm), and freeze-dried prior to ana-lysis The peptides were treated by acidolysis (6M HCl/ phenol) and their amino acid contents were determined (Beckman, System 6300 amino acid analyzer) The peptides were also analyzed by mass spectrometry (RP-DE Voyager, Perseptive Biosystems), and Edman sequencing using a gas-phase microsequencer (Applied Biosystems 470A)
In standard HPLC conditions for analyzing phenylthio-hydantoin (PTH) amino acid derivatives, diPTH-cystine eluted at a retention time of 9.8 min
Lethal activity of sPi4 in mice The sPi4 was tested in vivo for neurotoxicity by determining the 50% lethal dose (LD50) after intracerebroventricular injection of 20 g C57/Bl6 mice (animal testing agreement number 006573 granted by the department Sante´ et Protection Animales, Ministe`re de l’Agriculture et de la Peˆche) Groups of four to six mice per dose were injected with 5 lL sPi4 solution containing 0.1% bovine serum albumin and 0.9% sodium chloride (w/v)
Binding of sPi4 on SK channels from rat brain synaptosomes
Rat brain synaptosomes (P2 fraction) were prepared according to Gray and Whittaker [19] Protein content was assayed by a modified Lowry method [125I]Apamin (2000 CiÆmmol)1) was obtained as described [20] Aliquots
of 50 lL 0.1 nM [125I]apamin were added to 400 lL of synaptosome suspension (0.4 mg proteinÆmL)1) The sam-ples were incubated for 1 h at 0C with 50 lL of one of a series of concentrations of sPi4 or apamin (10)2)10)13M) in
500 lL final volume The incubation buffer was 25 mM
Tris/HCl, 10 mM KCl, pH 7.2 The samples were centri-fuged and the resulting pellets were washed three times with
1 mL of the same buffer Bound radioactivity was counted (Packard Crystal II) Reported values represent the means
Trang 3of triplicate experiments Nonspecific binding was evaluated
in the presence of an excess (10 nM) of unlabeled apamin
and was shown to be less than 8% of the total binding
Electrophysiology
Oocyte preparation Xenopus laevisoocytes (stages V and
VI) were recovered and prepared for cRNA injection and
electrophysiological recordings as described [21] The oocyte
follicular cell layers were removed by enzyme treatment
using 2 mgÆmL)1collagenase IA (Sigma) in classical Barth’s
medium [in mM: 88 NaCl, 1 KCl, 0.82 MgSO4,
0.33 Ca(NO3)2, 0.41 CaCl2, 2.4 NaHCO3,15
N-2-hydroxy-ethylpiperazine-N¢-ethanesulphonic acid (Hepes), pH 7.4
with NaOH] The plasmids were cleaved with SmaI
(Shaker B), Not1 (rat Kv1.1), Xba1 (rat Kv1.2) and EcoR1
(rat Kv1.3) The linearized plasmids were transcribed by
means of a T7 or SP6 mMessage mMachine kit (Ambion)
The cRNAs (1 lgÆlL)1) were kept frozen in H2O at)80 C
The cells were microinjected 2 days later with 50 nL of
cRNA (0.2 lgÆlL)1ShakerB, rat Kv1.1, Kv1.2, or Kv1.3
channels) To favor ion channel expression, cells were
incubated at 16C in a defined nutrient oocyte medium [22]
2 to 6 days before current recordings
Electrophysiological recordings A standard
two-micro-electrode technique was used to record oocyte currents (20–
22C) The current and voltage electrodes were filled with
140 mMKCl and had resistance values ranging between 0.5
and 1 MX The recordings of potassium currents were
performed using a voltage-clamp amplifier (GeneClamp
500, Axon Instruments, Foster City, CA, USA) interfaced
with a 16-bit AD/DA converter (Digidata 1200 A, Axon
Instruments) for acquisition and voltage protocol
applica-tion Current records were sampled at 10 kHz and low
pass-filtered at 2 kHz using an eight-pole Bessel filter The data
were stored on a computer for subsequent analysis
The extracellular recording solution contained (in mM):
88 NaCl, 10 KCl, 2 MgCl2, 0.5 CaCl2, 0.5 niflumic acid,
5 Hepes, pH 7.4 (NaOH) Leak and capacitive currents were
subtracted on-line by a P/4 protocol Residual capacitive
artifacts were blanked for display purposes The sPi4
solutions were perfused in the recording chamber at a flow
rate of 2 mLÆmin)1using a ValveBank4 apparatus
(Auto-mate Scientific Inc.) Bovine serum albumin (0.1%) was
added to the recording and perfusion solutions to prevent
toxin loss to the plastic chamber and tubules and nonspecific
binding onto the cell Data analysis was performed using the
softwarePCLAMP6.0.3 (Axon Instruments, Foster City, CA,
USA) Results are presented as mean ± SEM
Molecular modeling of rat Kv1 channel subtypes
Molecular modeling of rat Kv1 channel subtypes was based
on the crystal structure of KcsA solved at 2.0 A˚ resolution
(PDB accession number 1K4C) [23] The amino acid
sequences from residues 323–422 (corresponding to
domains S5–H5–S6) of the rat voltage-gated K+channel
a-subunits [24] were aligned with region 26–125 of KcsA
The sequence identities between the Kv-type ion channel
regions and KcsA are approximately 30% For the regions
between the two transmembrane segments S5 and S6, the
sequence identities with KcsA are approximately 50% Based on the high degree of similarity, the S5–H5–S6 regions of rat Kv1.1, Kv1.2, and Kv1.3 channels (Swiss-Prot accession numbers P10499, A33814 and P15384, respect-ively) were modeled by homology methods Sequences were aligned with clustal [25], the residue mutations were introduced in the KcsA channel structure with theTURBO
-FRODOsoftware [26], and the structures of the Kv channels thus obtained were minimized by using CNS [27] The final molecular models of the S5–H5–S6 regions of the rat Kv1.1, Kv1.2 and Kv1.3 channels adopt 3D structures which are similar to that of the KcsA channel (not shown) The transmembrane a-helices are comprised between residues 322–344 (S5) and 383–413 (S6) The pore region contains one a-helix between residues 360–370 Of note, the ion conducting pathway region is formed by four S5–H5–S6 domains The quality of all molecular models was assessed using the programsWHATIF[28] andPROCHECK[29] In each case, the stereochemical quality and the Ramachandran [30] scores were good and similar to that of the template Docking of Pi4 on rat Kv1.2 channel
The experimental 3D structure of sPi4 in solution, as recently solved by 1H-NMR [16], was used in docking experiments together with the molecular models we gener-ated for rat Kv channel subtypes (S5–H5–S6 domains) The molecular interaction simulations were performed using
BIGGER, a docking program [31] The algorithm used by
BIGGER performs a complete and systematic search for surface complementarity (both geometry complementarity and amino acid residue pairwise affinities are considered) between two potentially interacting molecules, and enables
an implicit treatment of molecular flexibility In each case,
a population of 1000 candidate protein–protein-docked geometries was selected byBIGGER In a subsequent step, the docked structures were ranked using an interaction scoring function, which combines several interaction terms that are thought to be relevant for the stabilization of protein complexes: geometric packing of the surfaces, electrostatic interactions, desolvation energy, and pairwise propensities
of the residue sidechains to contact across the molecular interface In the ab initio simulations, the entire molecular surface was searched using absolutely no additional infor-mation regarding the binding sites Among the 1000 candidate protein–protein-docked geometries selected, the five best scoring Pi4–Kv1.2 channel complexes were further treated with the TURBO-FRODO software, taking also into account the proposed functional maps of voltage-gated K+ channel-acting scorpion toxins [32–38] Finally, a rigid body minimization was used to minimize the selected complexes The best energy solutions, corresponding to the most favorable Pi4–Kv1.2 channel complexes, were selected The
de visu analysis was carried out using the TURBO-FRODO
software, and the geometric quality of the structures was assessed byPROCHECK3.3 [39]
Results and discussion
Pi4 (Fig 1) shares its highest sequence identities with scorpion toxins belonging to the a KTX6 subfamily: maurotoxin (68%) [3,4], Pi7 (66%) [1], Pi1 (61%) [5–7],
Trang 4and HsTx1 (45%) [8,9] This subclass of K+ channel
blockers contains short-chain toxins, from 34 to 38 amino
acid residues, cross-linked by four disulfide bridges (instead
of the three disulfide bridges generally observed in other K+
channel-acting scorpion toxins) To better characterize Pi4,
we chemically synthesized the toxin by using the solid-phase
technique [17]
Production of synthetic Pi4 (sPi4)
The Pi4 backbone assembly was achieved stepwise on
0.3 mmol HMP resin by means of an optimized Fmoc/t-Bu
chemical strategy [18] We found that the amount of target
peptide linked to the resin was 0.21 mmol, which indicates a
70% yield of peptide assembly Accordingly, a relative
homogeneity of the crude reduced peptide was observed
after final acidolytic treatment, as assessed by analytical C18
reverse-phase HPLC (Fig 2) The crude peptide was folded/
oxidized for 72 h in alkaline conditions using a standard
oxidative buffer [7], and the main oxidized species (sPi4) was
purified to >99% homogeneity by preparative C18
reverse-phase HPLC (Fig 2) An amino acid analysis of this
purified product showed amino acid ratios that were
consistent with the values deduced from the Pi4 primary structure (Fig 3) The mass spectrometry analysis (MALDI-TOF technique) of the peptide gave an experi-mental Mr(M + H)+of 4180.7, which is similar to the Mr
(M + H)+of 4180.9 calculated for Pi4 from its sequence The identity and homogeneity of sPi4 were also verified by Edman sequencing (data not shown) The total yield of sPi4 synthesis (which combines yields of peptide assembly, final acidolytic treatment, oxidative folding and peptide purifi-cation), was approximately 3% (9 lmol)
Disulfide bridge pattern of sPi4
In order to assign half-cystine pairings, sPi4 was treated by
a mixture of enzymes (trypsin and chymotrypsin) The resulting peptide fragments were separated by HPLC, then characterized by amino acid analysis, Edman sequencing and mass spectrometry (Fig 4) The results obtained from these techniques unambiguously map the half-cystine pair-ings as Cys6–Cys27, Cys12–Cys32, Cys16–Cys34 and Cys22–Cys37 Therefore, the disulfide bridge organization
of sPi4 is of the conventional type being C1–C5, C2–C6, C3–
C7and C4–C8, a pattern identical to that of Pi1 or HsTx1 but different from that of maurotoxin Of note, the disulfide bridges of natural Pi4 have been determined by NMR and structure calculations, as well as by Edman sequencing/mass spectrometry identification of peptides obtained by proteo-lysis of natural Pi4 [16] Both approaches have indicated half-cystine pairings between Cys6–Cys27, Cys12–Cys32, Cys16–Cys34 and Cys22–Cys37 for the natural toxin, consistent with the disulfide bridge arrangement found experimentally for sPi4 As expected, analysis of 2D1 H-NMR spectra of natural Pi4 and sPi4 [16] indicates that both peptides have the same structure
Biological properties of sPi4
In our bioassays, we tested sPi4 rather than its natural counterpart as the latter is present in too low abundance in the venom of scorpion P imperator to allow a detailed analysis of its structural and pharmacological properties
In vivo, sPi4 injected intracerebroventricularly produced
Fig 1 Amino acid sequence (one-letter code) of Pi4 and comparison
with other related scorpion toxin sequences The amino acid sequences
of Pi4 (P imperator) [1], maurotoxin (S maurus palmatus) [4], Pi7
and Pi1 (P imperator) [1,5–7], and HsTx1 (H spinnifer) [8] were
aligned according to the eight half-cystine residues The positions of
half-cystine residues are highlighted in gray boxes and numbered from
N- to C-terminus The asterisk indicates a C-terminal
carboxylami-dated extremity.
Fig 2 Analytical C 18 reverse-phase HPLC profiles of Pi4 at different stages of its chemical synthesis (A) The crude reduced peptide after final trifluoroacetic acid treatment (B) The crude peptide after 72 h folding/oxidation (C) The purified folded/oxidized peptide, sPi4 For conditions, see Experimental procedures.
Trang 5lethal effects in mice, with an LD50 value of 0.2 lg per
mouse This activity is identical to that of Pi1, but 2.5-fold
less potent than that of maurotoxin The lethal effects found
for sPi4 are about 10-fold less potent than those produced by
K+ channel-acting scorpion toxins reticulated by three
disulfide bridges [40,41] The sPi4-induced neurotoxic
symptoms resembled those of other K+ channel-acting
scorpion toxins suggesting that it targets some K+channels
In vitro, we first tested the ability of sPi4 to compete with
125I-labeled apamin for binding on rat brain synaptosomes
Figure 5 illustrates an sPi4-induced,
concentration-depend-ent, inhibition of125I-labeled apamin binding, with an IC50
value of 0.5 ± 0.2 lM At a similar concentration,
unlabe-led apamin produced a complete inhibition (IC100) of125
I-labeled apamin binding, with an IC value of 6 ± 3 pM
The half-effect of sPi4 occurred at a 100- or 10 000-fold higher concentration than that required for maurotoxin [3,4,42] or Pi1 [7], respectively, indicating a low, but significant, affinity interaction of sPi4 with rat brain apamin-sensitive SK channels Of note, HsTx1 was reported
to be inactive for binding on these SK channels [8], whereas Pi7 binding capability on the latter has not been described The blocking efficacy of sPi4 was also investigated by electrophysiology on different subtypes of K+channels that were heterologously expressed in Xenopus laevis oocytes
We focused on K+channels recognized by toxins from the
a KTX6 subfamily, i.e Shaker B, rat Kv1.1, Kv1.2 and Kv1.3 subtypes, and studied the putative sPi4-induced dose-dependent inhibition of currents associated with these channels Figure 6A shows that the application of 100 nM
sPi4 blocked over 80% of Shaker B currents The dose– response curves for sPi4 current inhibition were obtained for ShakerB (Fig 6B), rat Kv1.2 (Fig 6C), rat Kv1.1 and Kv1.3 channels (Fig 6D) The IC50 values of sPi4 were 3.0 ± 2.2 nMfor Shaker B (n¼ 45) and 8 ± 5 pMfor rat Kv1.2 (n¼ 55) channels, whereas it had no detectable effect
at concentrations up to 10 lMon rat Kv1.1 (n¼ 10) and Kv1.3 (n¼ 10) channels It is noteworthy that the maximal extent of blockage of K+ currents by sPi4 was approxi-mately 80% (Shaker B) or 60% (rat Kv1.2) of total currents Similar partial current blocks (from 50% to 80% of total currents) of these channels have been described for several scorpion toxins and their analogs, including Pi1 and maurotoxin [7,42–45] This phenomenon remains difficult
to explain, but an incomplete permeability block, possibly associated to an imperfect ion channel pore occlusion, can
Fig 4 Assignment of sPi4 half-cystine pairings (A) Characterization
of the sPi4-derived peptides that were generated by enzyme-based
cleavage of sPi4 (see Experimental procedures) Retention times in
HPLC (left column) and identified half-cystine pairings (right column)
of the peptides are shown (B) Complete disulfide bridge organization
of sPi4 as experimentally established by proteolysis of the synthetic
toxin The half-cystine residues are numbered according to their
positions in the Pi4 amino acid sequence The half-cystine connections
are represented by solid lines.
Fig 5 Binding of sPi4 on apamin-sensitive SK channels from rat brain synaptosomes Concentration-dependent inhibition of binding of [125I]apamin to rat brain synaptosomes by either unlabeled apamin (d)
or sPi4 (s) in a competition assay B 0 is the binding of [ 125 I]apamin without any other ligand, and B is the binding in the presence of the indicated concentrations of competitor Abscissa axis is the logarithm
of the molar concentration of competitor Nonspecific binding, less than 8% total binding, was subtracted for the calculation of ratios When absent, error bars are within symbol size The data were fitted to the equation y ¼ y o + a/[1 + exp(– (x ) IC 50 )/b)] The resulting
IC 50 values are 0.5 ± 0.2 l M (sPi4) and 6 ± 3 p M (unlabeled apamin).
Fig 3 Physicochemical characterization of sPi4 Amino acid content
(uncorrected values) of sPi4 after hydrolysis (118 C, 20 h, N 2
atmo-sphere) with 6 M HCl in the presence of 2% (w/v) phenol The deduced
amino acid composition is shown in parenthesis Deduced and
experimental relative molecular masses are indicated.
Trang 6tentatively be proposed Similar phenomena have also been
reported in a number of cases [42,43,45] From the
experimental data obtained both in vivo and in vitro, sPi4
appears to be pharmacologically more closely related to Pi1
than to maurotoxin, Pi7 or HsTx1 Indeed, the two toxins
apparently share the same lethal effects and selectivity profile
towards the tested K+channel subtypes (SK, Shaker B and
rat Kv1.2 channels), although their binding properties or
blocking efficacies towards these channels are clearly
distinct At the structural level, this should obviously rely
on marked differences of amino acid sequences between
both toxins, which guide the number and/or spatial
positioning of key functional residues that participate in
the interaction with the ion channel pore protein These data
strengthen the idea of a multipoint interaction between
scorpion toxins and their target ion channels As sPi4
blocked rat Kv1.2 channel at low picomolar concentrations,
we examined the interaction between Pi4 and this channel at
the molecular level, using the docking programBIGGER[31]
Docking experiments
To perform computed docking experiments, the structure of
sPi4 in solution recently solved from1H-NMR data [16] was
used, and specific models of rat voltage-gated K+channels
(S5–H5–S6 domains) [24] were generated According to
the docking simulation (Fig 7A–C), the toxin–ion channel
complex is stabilized by four salt bridges between the
sidechains of Glu332 of each rat Kv1.2 a-subunit
(Kv1.2 channel is composed of four a-subunits) and Arg10, Arg19, Lys30 and Lys33 of Pi4 The Lys26 sidechain
of Pi4 enters into the ion channel pore and is surrounded by the four Asp357 carbonyl oxygen atoms of the P-domain selectivity filter Residue Tyr35 of Pi4 is involved in a hydrophobic cluster of aromatic residues consisting of Trp344, from one of the four Kv1.2 a-subunits, and Trp345 and Tyr355 of an adjacent a-subunit The phenol ring of Tyr35 additionally forms an hydrogen bond with the Neof the Trp344 indole ring Some hydrophobic interactions are also likely to occur between Ile28 of Pi4 and Val361 of the Kv1.2 a-subunit For comparison, Pi1 was also docked on the Kv1.2 channel (data not shown) Similar types of low-energy interactions were found but involving Arg5, Arg12, Lys24 (dyad), Ile26, Arg28, Lys31 and Tyr33 (dyad) residues of Pi1 However, in the case of Pi1, toxin positioning over the channel was different, with a slight rotation over the channel as compared to Pi4, its Tyr33 being in contact with the cluster of aromatic residues belonging to the same Kv1.2 a-subunit Of note, Pi4 (or Pi1) did not give good scores when assayed for docking on rat Kv1.1 and Kv1.3 channels (data not shown), in agreement with its lack of bioactivity on these channels
Functional maps of Pi4 and Pi1 towards rat Kv1.2 channels
Results from docking experiments allow us to propose functional maps for both Pi4 and Pi1 regarding their
Fig 6 Blocking efficacy of sPi4 towards the voltage-gated K + channel subtypes (A) Oocytes expressing Shaker B K+channels were recorded under two-electrode voltage clamp K + currents were obtained by depolarization from a holding potential of )90 mV to +70 mV Left panel: Shaker B
K + control currents during superfusion of 100 n M of sPi4 illustrating over 80% block; right panel: K + currents during superfusion of 100 n M sPi4, illustrating over 60% block The dose–response curves for sPi4 current inhibition were performed for: (B) Shaker B, (C) rat Kv1.2, and (D) rat Kv1.1 (s) and Kv1.3 (d) channels The solid lines through the data are obtained from the equation y ¼ y o + a/[1 + exp(– (x ) IC 50 )/b)] The
IC 50 values of sPi4 were 3.0 ± 2.2 n M for Shaker B (n ¼ 45) and 8 ± 5 p M for rat Kv1.2 (n ¼ 55) channels No significant effects on rat Kv1.1 (n ¼ 10) and Kv1.3 (n ¼ 10) channels were detected at sPi4 concentrations up to 10 l M Data points are the mean ± SEM When absent, error bars are within symbol size All inhibitions were determined by inducing currents by depolarizations at +70 mV.
Trang 7recognition and blockage of rat Kv1.2 channels Fig 8A,B).
These maps suggest an important contribution of Arg10,
Arg19, Lys26, Ile28, Lys30, Lys33 and Tyr35 residues for
Pi4, as well as of Arg5, Arg12, Lys24, Ile26, Arg28, Lys31
and Tyr33 residues for Pi1 The functional dyads are
attributed to Lys26 and Tyr35 for Pi4 [1], and Lys24 and
Tyr33 for Pi1 [7] Therefore, as mentioned by
Olamendi-Portugal et al [1], the substitution of the functional Lys26
in Pi4 for an Arg26 in the structurally homologous Pi7
might be a key natural point mutation responsible for the
lack of activity of Pi7 on Kv channels We suggest a
two-step pictorial view of Pi4 binding in which the toxin ring of
basic residues (ring composed of Arg10, Arg19, Lys30 and
Lys33) plays a crucial role (via electrostatic forces) in the
recognition, interaction and correct positioning of Pi4 on
the Kv1.2 channels, and then a tighter interaction takes
place through both hydrophobic forces and hydrogen
bonding between Tyr35 (dyad) and the aromatic cluster
consisting of Trp344, Trp345* and Tyr355* (Fig 7B,
legend), and between Ile28 and Val361 The Lys26 (dyad)
sidechain enters the ion channel pore and is stabilized by
the four Asp357 carbonyl oxygen atoms of the Kv1.2
a-subunits; the Lys sidechain presumably acts by blocking
K+ion flux through the pore, and might thus be involved
in the toxin blocking efficacy
Presence of the ring of basic residues in other toxins active on Kv1.2 channels
To examine the potential importance of the ring of basic residues in the recognition and interaction of Pi4 with Kv1.2 channels, we focused on two scorpion toxins, Pi2 (P im-perator) [6,46] and TsTXa (Tityus serrulatus) [47], that are also classified as high affinity blockers of Kv1.2 channels (both being active at the picomolar concentration range) and of known 3D structures [48,49] The two toxins possess well-defined b-sheet-associated functional dyads, i.e Lys27 and Tyr36 (TsTXa) and Lys24 and Phe33 (Pi2) Of note, the usual aromatic Tyr is replaced by an aromatic Phe in the case of Pi2, which is thought to interact, via its phenyl ring, with the aromatic cluster of the Kv1.2 a-subunit as well Pi2 and TsTXa also exhibit a four-membered ring of basic residues similar to that of Pi4 or Pi1 It is clear that more structure–activity relationship studies on these toxins are needed to validate the idea of a possible key role of such a ring in the toxin binding on Kv1.2 channels
Conclusions
From a number of previous reports on different scorpion toxins that act on Kv-type channels, it appears that the toxin
Fig 7 Docking of sPi4 on rat voltage-gated Kv1.2 channel (pore region) (A) Side view ( TURBO - FRODO software) depicting the interaction of sPi4 [1] (structure solved by 1 H-NMR) with rat voltage-gated Kv1.2 channel (molecular model of the S5–H5–S6 domains) [24] For clarity, Ca peptide backbones of only two out of the four S5–H5–S6 a-subunits of the Kv1.2 channel are presented (deep blue) The Ca peptide backbone of sPi4 is shown in green Only the sidechains of amino acid residues that are involved in the sPi4–Kv1.2 channel interaction are displayed Basic, acidic and aromatic residues are shown in light blue, red and purple, respectively The residues are numbered according to their positions within the Pi4 and rat Kv1.2 a-subunit amino acid sequences [1,24] (B) Magnified side view showing the interactions of sPi4 with the rat Kv1.2 channel For sPi4, only the sidechains of residues involved in this interaction are depicted Also, only interacting residues from the Kv1.2 a-subunits are pictured in their exact 3D positions, according to the ion channel molecular model (see Fig 7A for details) The asterisks indicate that the corresponding residues belong
to distinct a-subunits (C) Top view showing the docking of sPi4 on rat voltage-gated Kv1.2 channel (pore region) Only interacting residues are presented with their corresponding sidechains (see Fig 7A for details) The four a-subunits (S5–H5–S6 domains) forming the Kv1.2 channel are noted from A to D.
Trang 8b-sheet structure plays a premium role in binding to these
channels [32,41,50,51] Amongst the residues belonging to
the b-sheet, the key contribution of a pair of well-defined
basic and aromatic residues, referred to as the functional
dyad, which we attributed to Lys26 and Tyr35 in the case of
Pi4, has been shown The docking of Pi4 (or Pi1) on rat
Kv1.2 channels further provides additional insights into the
structural basis of this recognition/interaction Indeed, an
unexpected contribution of a ring composed of four basic
residues belonging to various faces of the toxin has been
highlighted, which supports the idea of a multipoint
interaction between Pi4 and this ion channel It is interesting
to note that this ring of basic residues also exists in other
potent Kv1.2 channel-acting scorpion toxins, such as Pi1 [7],
Pi2 [6,46], and TsTXa [47,49] At the level of rat Kv1.2
channel, a key functional residue appears to be Glu332 of the
a-subunit, a residue absent in Kv1.1 and Kv1.3 a-subunits
[24] In the context of the channel, the four Glu332 from the
four a-subunits are thought to interact, via salt bridges, with
the four residues from the toxin ring of basic residues The
production of some selected Pi4 analogs, notably those with
an altered ring of basic residues, will help to test
experi-mentally the Pi4 functional map deduced from the docking
experiments Because the latter also gave some insights that
might potentially explain the selectivity of the Pi4 action on
voltage-gated Kv1.2 channels, the docking approach will be
used to design Pi4 analogs that exhibit some changes in
pharmacological selectivity or affinity towards the K+
channel subtypes The completeness of Kv1.2 pore occlusion
by Pi4 is a parameter that can tentatively be improved by
selective mutation of some Pi4 residues It is worth noting
that the actual docking simulation of Pi4 is informative but
remains insufficient to reasonably explain, at a molecular
level, the partial Kv1.2 pore occlusion The present study will
be further extended to other Kv-channel acting scorpion toxins [6,9,47,52] to shed light on the molecular basis of the toxin to Kv-type channel recognition Finally, it will be interesting to test the sPi4 bioactivity on different classes of
K+channels (e.g Eag, HERG, KCNQ, Slo, IKCa, Kir, and KCNK) in order to determine the uniqueness or not of toxin action on Kv1.2 channel
Acknowledgements
The authors wish to thank Drs J Van Rietschoten and C Devaux for helpful discussions This work was supported by funds from the CNRS and Cellpep SA (Paris, France) Dr A Mosbah is a recipient of a fellowship from Cellpep SA.
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