Recombinant PBTx3 inhibits both Kv1.2 and Kv1.3 channels with weak affinities and similar potencies, whereas it is a very weak inhibitor of Kv1.1 channels: application of 550 nM rPBTx3 p
Trang 1Purification, characterization and biosynthesis of parabutoxin 3,
Isabelle Huys1, Karin Dyason2, Etienne Waelkens3, Fons Verdonck4, Johann van Zyl5, Johan du Plessis2, Gert J Mu¨ller5, Jurg van der Walt2, Elke Clynen6, Liliane Schoofs6and Jan Tytgat1
1
Laboratory of Toxicology, University of Leuven, Leuven, Belgium;2Department of Physiology, University of Potchefstroom, Potchefstroom, South Africa;3Laboratory of Biochemistry, University of Leuven, Leuven, Belgium;4Interdisciplinary Research Centre, University of Leuven Campus Kortrijk, Kortrijk, Belgium;5Department of Pharmacology, University of Stellenbosch, Tygerberg, South Africa;6Laboratory for Developmental Physiology and Molecular Biology, University of Leuven, Belgium
A novel peptidyl inhibitor of voltage-gated K+channels,
named parabutoxin 3 (PBTx3), has been purified to
homo-geneity from the venom of Parabuthus transvaalicus This
scorpion toxin contains 37 residues, has a mass of 4274 Da
and displays 41% identity with charybdotoxin (ChTx, also
called Ôa-KTx1.1Õ) PBTx3 is the tenth member (called
Ôa-KTx1.10Õ) of subfamily 1 of K+channel-blocking
pep-tides known thus far Electrophysiological experiments using
Xenopus laevisoocytes indicate that PBTx3 is an inhibitor of
Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable
effects on Kir-type and ERG-type channels The
dissoci-ation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels
are, respectively, 79 lM, 547 nM and 492 nM A synthetic
gene encoding a PBTx3 homologue was designed and
expressed as a fusion protein with the maltose-binding
pro-tein (MBP) in Escherichia coli The recombinant propro-tein was
purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa(fXa) to release the recombinant toxin peptide (rPBTx3) After final purifi-cation and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins
Keywords: Parabuthus; purification; synthesis; scorpion; toxin
The southern African scorpion Parabuthus transvaalicus
Purcell, 1899, is one of the largest scorpions belonging to the
Buthidae family [1], subphylum Chelicerata, order
Scor-pionis Severe envenomation with P transvaalicus causes
primarily neuromuscular effects with involvement of the
heart and parasympathetic nervous system [2], illustrating
that this scorpion can be potentially lethal, especially for
children P granulatus scorpionism has been described by
Mu¨ller [3] P transvaalicus scorpionism is clinically similar,
but appears to produce slightly more motor and fewer
sensory symptoms [4] Crude, diluted venom of P
trans-vaalicuswas already tested on isolated cardiomyocytes and
induced an increase in the sodium current and a retardation
of the time course of inactivation, implicating the presence
of an a-toxin [5] Verdonck et al [6] reported the occurrence
of pore-forming activity in the venom of P transvaalicus,
but the variability was rather high and in some specimens this activity was absent
A study was undertaken to find compounds or toxins in the venom of P transvaalicus that modulate physiological processes at the cellular level; this was done for the following reasons: (a) very little is known about the bioactive substances present in the venom of this scorpion [7,8]; (b) the discovery of new toxins can be the key to gain insight into the molecular mechanisms of scorpionism; (c) selective toxins can be used for purifying channels from native tissue, determining their subunit composition [9] and for elucida-ting the pharmacology and physiological roles of voltage-dependent Na+, Ca2+and K+channels [10–12] in target tissues Voltage-dependent K+channels in particular serve important functions in many signal-transduction pathways
in the nervous system: they are involved in neuron excitability; they influence the resting membrane potential, the waveforms and frequencies of action potentials; and they determine the thresholds of excitation [13] Moreover, they are the putative target sites in the design of therapeutic drugs [14]
In our work, a new short-chain toxin acting on Kv1 channels, called parabutoxin 3 (PBTx3), has been purified
to homogeneity from the venom of P transvaalicus and its specific function on different channels has been analysed electrophysiologically Using a recombinant expression system, the toxin was produced in high quantity to confirm our data and to facilitate the screening of the active peptide
Correspondence to J Tytgat, Laboratory of Toxicology, University
of Leuven, E Van Evenstraat 4, 3000 Leuven, Belgium.
Fax: + 32 16 32 34 05, Tel.: + 32 16 32 34 03,
E-mail: Jan.Tytgat@farm.kuleuven.ac.be
Abbreviations: PBTx3, toxin from the venom of the scorpion
Parabuthus transvaalicus; AgTx2, toxin from the venom
of the scorpion Leiurus quinquestriatus var Hebraeus; MBP,
maltose-binding protein; fXa, factor Xa.
Note: a website is available at http://www.toxicology.be
(Received 31 December 2001, accepted 12 February 2002)
Trang 2PBTx3 In this way, a study of the structure–function
relationship of PBTx3 to different ion channels and
receptors could be performed and a structural model for
this novel toxin has been proposed
M A T E R I A L S A N D M E T H O D S
Venom collection and purification
P transvaalicusscorpions were captured in South Africa
Venoms were collected by electrical stimulation and
lyophi-lized after dilution in a saline buffer or distilled water The
lyophilized venom was dissolved in 100 mM ammonium
acetate, pH 7 (Merck, Germany) After vortexing, the
sample was clarified by centrifugation at 12 000 g for
15 min and its supernatant was submitted to gel filtration
(Fig 1A) using a Superdex 30 prep grade HiLoad 16/60
FPLC column (Pharmacia LKB Biotech, Sweden)
equili-brated with 100 mMammonium acetate, pH 7 The
mate-rial was eluted with the same buffer at a flow rate of 0.2 mLÆmin)1 Absorbance of the eluate was monitored at
280 nm and 4-mL fractions were collected automatically The fraction containing the toxin was recovered, lyophilized and applied on a PepRPC HR 5/5 C2/C18reversed-phase FPLC column (Pharmacia, Sweden) equilibrated with 0.1% trifluoroacetic acid (TFA, Merck Eurolab, Belgium) in distilled water (Fig 1B) Separation was performed by using
a linear gradient of 0–50% UV-grade acetonitrile (LiChro-SolvÒ gradient grade, Merck Eurolab), supplemented with 0.1% TFA, for 30 min The flow rate was 0.5 mLÆmin)1 and the absorbance was measured at 214 nm Fractions between 17 and 23 min with potential short-chain toxins were recovered, dried (Speed VacÒ Plus, Savant, USA), and applied to a monomeric 238TP54 C18reversed-phase HPLC column (Vydac, USA) equilibrated with 0.1% trifluoroacetic acid in distilled water (Fig 1C) Separation was performed
as follows: after 4 min a linear gradient to 30% acetonitrile, for 2 min, followed by a linear gradient to 42% for the final
8 min (total run, 14 min) The flow rate was 0.75 mLÆmin)1 and the absorbance was measured simultaneously at 214,
254 and 280 nm The toxin-containing fraction (see Fig 1C) was recovered and dried (Speed VacÒ Plus)
Sequence determination The first 36 residues of the primary structure of the peptide were resolved by direct sequencing (Edman degradation) (Fig 2A) A glass fibre disk was coated with Biobrene (Applied Biosystems) and precycled for four cycles Subse-quently, the sample (18 pmol) was loaded onto the glass fibre disk and subjected to N-terminal amino-acid sequenc-ing on a Perkin Elmer/Applied Biosystems Procise 492 microsequencer (PE Biosystems) running in pulsed liquid mode To identify the last C-terminal residue, a sample of peptide was also cleaved by cyanogen bromide By this reaction, three fragments were produced (E1–M4, R5–M28 and N29–R37), separated by HPLC by using the same C18 analytical column as described above, and then sequenced The last amino acid (arginine) was elucidated
Construction of the recombinant genes
A cDNA fragment encoding a 36 amino-acid peptide, corresponding to PBTx3 without the C-terminal arginine, was designed as follows (Fig 3A) Two overlapping oligonucleotide pairs 5¢-GAGGTCGACATGCGCTGCA AGTCGTCGAAGGAGTGCCTGGTCAAGTGCAAG CAG-3¢, 3¢-CTCCAGCTGTACGCGACGTTCAGCAG CTTCCTCACGGACCAGTTCACGTTCGTCCGCTG CCCGGCC-5¢, and 5¢-GCGACGGGCCGGCCGAACG GCAAGTGCATGAACCGGAAGTGCAAGTGCTAC CCGTGAG-3¢, 3¢-GGCTTGCCGTTCACGTACTTGGC CTTCACGTTCACGATGGGCACTCCTAG-5¢, respect-ively, ranging in length from 49 to 66 base pairs, were synthesized chemically on an Applied Biosystem device (Amersham Pharmacia Biotech, The Netherlands), purified
by PAGE and phosphorylated at the 5¢ end The comple-mentary oligomers (100 pmol of each) were annealed to generate two duplexes that were ligated using T4 DNA ligase (NEB) The synthetic PBTx3 gene was inserted into the vector pMAL-p2X (NEB) downstream from the malE gene
of Escherichia coli and also directly downstream of a fX site
Fig 1 Purification of native PBTx3 from the venom of P
transvaali-cus (A) Crude venom was first fractionated by FPLC gel filtration,
yielding four peaks The labelled fraction (*) was recovered and
lyo-philized Based on a constructed gel filtration calibration curve, the
molecular mass of the material in this fraction ranged from 3 to 6 kDa.
(B) The second purification step was carried out using a FPLC C 2 /C 18
reversed-phase column Fractions eluting at 17–23 min (*) contain
ÔpotentialÕ short-chain toxins and were recovered and dried (C) The
third step involved a HPLC C reversed-phase purification.
Trang 3into a XmnI site The gene possessed an overhang at the
3¢ end (BamHI) to direct the orientation of the insert into
pMAL-p2X The transformants containing the correctly
constructed DNA fragments for PBTx3 were analysed by
digestion with two different restriction enzymes NaeI and
XmnI (NEB) Because insertion of the synthetic gene disrupts
the XmnI recognition site, this enzyme cannot cleave the
recombinant plasmid To cleave the gene in the second part
of its sequence, NaeI was used as a double control of the
original duplexes In both cases, E coli JM109 (Promega,
The Netherlands) was used for plasmid propagation A
translation termination codon was inserted at the end of the
PBTx3 coding sequence The vector possesses malE
trans-lation initiation signals to direct the toxin-fusion proteins to
the periplasm, thus allowing folding and disulfide bond
formation to take place in E coli [15,16] The method for the
expression of our toxins used the strong Ptacpromoter, which
gave a high-level expression of the cloned sequences encoding
the fusion For comparison with PBTx3, the high affinity K+
channel blocker AgTx2 [17], which is structurally related to
PBTx3, was produced by a similar strategy
Expression, purification and cleavage
of fusion proteins
Rich Luria–Bertani medium containing bactotryptone
(Sigma, Belgium), yeast (Remel, BioTrading, Belgium),
NaCl (Merck Eurolab, Belgium), glucose (Merck Eurolab) and ampicillin (1 lgÆmL)1) was inoculated with an over-night culture of E coli DH5a cells, carrying the gene fusions encoding either rAgTx2 or rPBTx3, in a culture shaker incubator (Innova 4000, New Brunswick Scientific) In both situations, the cells were grown at 37°C and when the cell density had reached A600 ¼ 0.5, expression of the fusion proteins was induced by adding isopropyl thio-b-D -galacto-side (Sigma) to a final concentration of 0.2 mM Cells were harvested by centrifugation at 2660 g at 4°C for 20 min and subjected to osmotic shock according to the following procedures The cells were resuspended in 400 mL 30 mM Tris/HCl (Sigma) with 20% sucrose (Sigma) pH 8.0 at
25°C The suspension was treated with Na2EDTA (Sigma)
to give a concentration of 1 mM and incubated at room temperature with shaking After 10 min, the mixture was centrifuged for 20 min at 2660 g at 4°C The supernatant was removed and the well drained pellet was resuspended in
400 mL ice-cold 5 mMMgSO4(Sigma) in an ice bath for
10 min and centrifuged at 2660 g at 4°C The supernatant
is the cold osmotic shock fluid which contains the periplas-mic extracts The periplasperiplas-mic extracts (400 mL) were loaded
Fig 2 Sequence determination of native PBTx3 (A) The first 36
amino acid residues of PBTx3 were identified by direct sequencing a
Sequencing the last fragment, produced after cyanogen bromide
cleavage, identified the C-terminal residue arginineb (B) Alignment of
the amino acid sequences of the members of subfamily 1 of short-chain
a-KTx toxins isolated from scorpion venom Dashes represent gaps
that were introduced to improve the alignment Identical amino acids
are indicated with a black background Homologous residues are
indicated with a grey background The percentage identity with ChTx
is shown ChTx (charybdotoxin [24]), charybdotoxin-Lq-2 [10], Lqh
15–1 [25] and AgTx2 (agitoxin 2 [15]), were purified from Leiurus
quinquestriatus var Hebraeus; BmTx1–2 [26] was purified from Buthus
martensi Karsch; HgTx2 (hongotoxin 2 [27]), and LbTx (limbatotoxin
[34]), were purified from Centruroides limbatus; IbTx (iberiotoxin [28]),
and TmTx (tamulotoxin [56]), were purified from Buthus tamulus;
PBTx3 (parabutoxin 3, this study) was purified from Parabuthus
transvaalicus.
Fig 3 Schematic diagram of the pMAL-p2X vector containing the synthetic gene for the PBTx3 homologue (A) Two ligations were performed using a 6706-bp pMAL-p XmnI/BamHI fragment and a 111-bp fragment encoding the PBTx3 homologue, immediately downstream of the fX a cleavage site in the vector Amp R , ampicillin resistance gene; ori, origin (B–D) Chromatographic profiles after purification of the fusion protein (B) and recombinant toxin (C,D) rPBTx3 Fractions containing the MBP-fusion proteins were collected and prepared for cleavage with fX a The restriction digests were applied on the same HPLC C 18 column as in Fig 1 and material eluting between 8 and 15 min was purified further on a HPLC C 2 /C 18 column and tested on Kv1 channels expressed in Xenopus oocytes.
Trang 4to an amylose affinity resin (1.5· 23 cm column, Biolabs,
NEB) at a flow rate of 1 mLÆmin)1 in column buffer
containing 20 mM Tris/HCl, 200 mM NaCl (Merck
Eur-olabs, Belgium), and 1 mMNa2EDTA buffer, pH 7.4 After
washing of the unbound proteins, the bound
maltose-binding protein (MBP)-fusion products were eluted from
the amylose resin using the same column buffer containing
10 mMmaltose (Merck Eurolabs) Twenty 3-mL fractions
were collected and the fusion protein was easily detected by
the UV absorbance spectrophotometer (UV/VIS
Spectro-photometer lambda 16, PerkinElmer) at 280 nm The
protein-containing fractions were pooled and purified
further using a Superdex Peptide gel filtration column on
the SMART System (Pharmacia Biotech) The elution was
performed with a buffer containing 20 mM Tris/HCl and
100 mMNaCl, pH 8.0 (Fig 3B) Controls were performed
with cells containing no vector or cells containing the vector
without insert
The synthetic gene encoding the PBTx3 homologue was
designed such that an fXacleavage site (Ile-Glu-Gly-Arg-)
immediately preceded the N-terminal Glu of the toxin
(Fig 3A) The enzymatic cleavage of the pooled fusion
proteins was carried out at various conditions by fXa
(different sources: Boehringer, Sigma, NEB) Optimal
cleavage could be performed in the following conditions:
72 h incubation at room temperature and a concentration
of 0.5 UÆlg)1fusion protein in a buffer containing 20 mM
Tris/HCl, 100 mM NaCl and 2 mM CaCl2, pH 8.0 After
cleavage with this enzyme, the recombinant toxin was
generated without vector-related fragments In a parallel
experiment with AgTx2, chromatographic profiles of
rAgTx2 and commercially available rAgTx2 (Alomone
Laboratories) under the same conditions were compared
and were identical
HPLC
Separations of the recombinant proteins were first
per-formed with a 218TP104 C18reversed-phase HPLC column
(Vydac) and equilibrated with 0.1% trifluoroacetic acid
(Sigma) at 25°C (Fig 3C) After 4 min an immediate step
to 5% acetonitrile (with 0.1% trifluoroacetic acid) was
followed by a linear gradient to 30% acetonitrile for 5 min
and then by a linear gradient to 60% for the last 12 min
The flow rate was 0.75 mLÆmin)1and the absorbance was
measured simultaneously at 214, 254 and 280 nm The
fraction containing the recombinant toxin (arrow) was
recovered and applied to a lRPC C2/C18 SC 2.1/10
reversed-phase HPLC column (Vydac) A linear gradient,
starting after 6 min and ranging from 0% to 30% up to
100 min with a flow rate of 200 lLÆmin)1(Fig 3D), was
applied and the toxin was collected, dried (Speed VacÒ
Plus) and prepared for functional analysis
Mass spectroscopy
For examination of mass, 1 pmol of the venom was dried
and redissolved in acetonitrile (+ 0.1% trifluoroacetic
acid) The molecular mass of the compounds in the venom
and the masses of rAgTx2 (used as a control toxin) and
rPBTx3 were determined with MALDI-TOF MS on a VG
Tofspec (Micromass, UK) operating in the linear and in the
reflectron mode
Electrophysiological recording Oocyte expression – Kv1.1 For in vitro transcription, plasmids were first linearized with PstI (New England Biolabs) 3¢ to the 3¢ nontranslated b-globin sequence in our custom-made high expression vector for oocytes, pGEMHE [18–20] and then transcribed using T7 RNA polymerase and
a cap analogue diguanosine triphosphate (Promega) Kv1.2 The cDNA encoding Kv1.2 (originally termed RCK5) in its original vector, pAKS2, was first subcloned into
pGEM-HE [19] The insert was released by a double restriction digest with BglII and EcoRI Next, the cDNA was loaded onto an agarose gel, fragments of interest were cut out, gene cleaned (QIAGEN) and ligated into the BamHI and EcoRI sites of pGEM-HE For in vitro transcription, the cDNA was linearized with SphI and transcribed using the large-scale T7 mMESSAGE mMACHINE transcription kit (Ambion) Kv1.3 Plasmid pCI.neo containing the gene for Kv1.3 was linearized with NotI (New England Biolabs) and transcribed as for Kv1.2 [21] Stage V–VI Xenopus laevis oocytes were isolated by partial ovariectomy under anaes-thesia (tricaine, 1 gÆL)1) Anaesthetized animals were kept
on ice during dissection The oocytes were defolliculated by treatment with 2 mgÆmL)1 collagenase (Sigma) in zero calcium ND-96 solution (see below) Between 2 and 24 h after defolliculation, oocytes were injected with 50 nL of 1–
100 ngÆlL)1cRNA The oocytes were then incubated in ND-96 solution at 18°C for 1–4 days The animals were handled in conformity with the ‘Guide for the Care and Use
of Laboratory Animals’, published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996)
Electrophysiology Whole-cell currents from oocytes were recorded using the two-microelectrode voltage clamp technique Voltage and current electrodes (0.4–2 mega-ohms) were filled with 3M KCl Current records were sampled at 0.5-ms intervals after low pass filtering at 0.1 kHz Off-line analysis was performed on a Pentium(r) III processor computer Linear components of capacity and leak currents were not subtracted All experiments were performed at room temperature (19–23°C) Fitted Kd values were obtained after calculating the fraction current left over after application of several toxin concentrations in different oocyte experiments (mean ± SD, n)
Solutions The ND-96 solution (pH 7.5) contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, supplemented with 50 mgÆL)1gentamycin sulphate (only for incubation)
Modeling
A model was generated by an automated homology modelling server (Expert Protein Analysis System proteo-mics server using SWISS-MODEL-ProModII) running at the Swiss Institute of Bioinformatics (Geneva) Target (PBTx3) and template (hongotoxin 2) sequences were automatically aligned by Multiple Sequence Alignment Software (CLUSTALW), which subsequently generated the coordinates of both models Energy minimization (GROMOS96) and simulated annealing cycles were run
- computes a confidence factor for each atom
Trang 5in the model structure, taking into account the deviation of
the model from the template structure and the distance trap
value used for framework building
R E S U L T S
Kv1 K+channels were expressed in X laevis oocytes and
studied using a two-microelectrode voltage clamp Crude
venom of P transvaalicus (340 lg) produces a reversible
inhibition of the Kv1.1 K+current elicited by
depolariza-tion up to 0 mV (data not shown) In our quest to find novel
short-chain scorpion toxins in the venom of P
transvaali-cus, acting on voltage-dependent K+channels, we
fraction-ated the crude venom of this scorpion as detailed in
Materials and methods (Fig 1) As described by Debont
et al [22], gel filtration shows three typical groups of
components (Fig 1A), the largest of which (group I) was
shown to block Kv1.1 channels Based on a constructed
calibration curve (see Debont et al 1998), the active fraction
corresponded to a molecular mass between 3 and 6 kDa,
which probably represents the family of short-chain
scor-pion toxins After HPLC purification of this active fraction
(Fig 1C), that representing native PBTx3 (85 lM) caused
an inhibition of Kv1.1 channels of 50%, whereas 550 nM
native PBTx3 produces 54% and 51% block of the Kv1.2
and Kv1.3 channels, respectively (Fig 4A–C) We have
undertaken the recombinant synthesis of this toxin in order
to facilitate the characterization of its biological properties
The yields of affinity-purified proteins were 40–60 mgÆL)1
culture, estimated by absorbance at 280 nm, which after
cleavage resulted in the production of 2–4 mg of
recombin-ant toxin per litre culture The recombinrecombin-ant synthesis
resulted in the production of a recombinant toxin with an
expected molecular mass of 4118 Da, with respect to the
three disulfide bridges present in the secondary structure of
the PBTx3 homologue The mass of rAgTx2 (ÔcontrolÕ toxin
for comparison) was also consistent with the theoretical
mass Functional effects of recombinant toxins on Kv.1
channels were investigated by electrophysiological
experi-ments No block was obtained when MBP-rPBTx3 was
applied to expressed K+ channels in Xenopus oocytes
(n ¼ 3) (data not shown) Recombinant PBTx3 inhibits
both Kv1.2 and Kv1.3 channels with weak affinities and
similar potencies, whereas it is a very weak inhibitor of
Kv1.1 channels: application of 550 nM rPBTx3 produced
no blocking effect on Kv1.1 channels (Fig 4D), whereas the
Kv1.2 and Kv1.3 currents were reversibly blocked to 52%
and 49%, respectively (Fig 4E,F) As part of a control,
rAgTx2 was applied to the same oocytes expressing Kv1.1
channels Addition of 1 nMrAgTx2 blocked the K+current
almost completely (Fig 4G) and this effect was reversible
upon washout After equilibration of the channels and
application of the same concentration of commercially
available rAgTx2, quantitatively the same effect was
observed as with our laboratory prepared rAgTx2 This
observation, together with the fact that co-injection of
equimolar amounts of both AgTx2 on reverse-phase HPLC
resulted in a single peak (data not shown), demonstrates
that our rAgTx2 behaved similarly to the commercially
available recombinant toxin
Blockage of the Kv1 channels induced by rAgTx2 or
rPBTx3 (tested at different concentrations) was shown not
to be voltage-dependent, as the degree of block was not
different in the range of test potentials from )30 to +20 mV Recombinant PBTx3 (500 nM) blocked the Kv1.2 and Kv1.3 peak currents by 54% and 53% at )30 mV (n ¼ 3), and by 53% and 54% (n ¼ 3) at
20 mV In the presence of 70 lMrPBTx3, the Kv1.1 peak current was blocked by 45% (n ¼ 3) at)30 mV and by 42% at 20 mV (n ¼ 3)
Blocking of the Kv1 channels by rPBTx3 is reversible and has no influence on the gating characteristics of the channels Therefore, the time constants for relaxation to equilibrium block of the different Kv1 channels in the presence of the toxin reflect only the progress of the binding reaction To determine the time constants sonand sofffor blockade and recovery, current traces were repeated every
2 s before, during and after rPBTx3 application The time-courses of blockade and recovery were fitted to mono-exponential curves, in agreement with the results obtained for other scorpion toxins [23] In the presence of 10 lM rPBTx3 on Kv1.1 and 3.3 lMrPBTx3 on Kv1.2 and Kv1.3 channels, blockade occurred with a mean time constant son
of 8.3 ms, 2.1 ms and 1.7 ms, respectively, for Kv1.1, Kv1.2 and Kv1.3 channels The recovery from blockade
Fig 4 Effects of native (A–C) and recombinant (D–F) PBTx3 on Kv1.1, Kv1.2 and Kv1.3 channels Whole-cell K+ currents through Kv1.1, Kv1.2 and Kv1.3 channels, respectively, expressed in Xenopus oocytes, are evoked by depolarizing the oocyte from a holding potential of )90 mV to 0 mV The oocytes were clamped back to )90 mV (A), or to )50 mV (B–G) Application of 85 l M native PBTx3 (active fraction in Fig 1C indicated by *) on Kv1.1 channels or 550 n M
on Kv1.2 and Kv1.3 channels, produced 50%, 54% and 51% inhibi-tion, respectively, of the Kv1.1, Kv1.2 and Kv1.3 currents (D–F) Current through Kv1.1, Kv1.2 and Kv1.3 channels, respectively, in control conditions (s) and in the presence (d) of 550 n M rPBTx3 (G) Inhibition of Kv1.1 current, produced by 1 n M of rAgTx2.
Trang 6occurred with a mean soffof 9.2 ms, 23.9 ms and 10.8 ms.
Corresponding konvalues were therefore 1.3· 103
M )1Æs)1, 12.7· 104
M)1Æs)1 and 1.4· 105
M )1Æs)1 and koff values were 0.108 s)1, 0.041 Æs)1 and 0.092 Æs)1, respectively, for
Kv1.1, Kv1.2 and Kv1.3 The Kdcalculated from the ratio
koff/konwas in all cases in good agreement with the value
obtained in the dose–response experiments (see further):
80 lM for Kv1.1 (Kd ¼ 79 lM), 322 nM for Kv1.2
(Kd ¼ 547 nM) and 657 nM for Kv1.3 (Kd ¼ 492 nM)
The fraction of unblocked current at equilibrium (fu) is
readily measured and is related to the rate constants
according to fu ¼ koff/(kon[rPBTx3] + koff) The Hill
coefficients were not significantly different from 1 From
the constructed current/voltage relationship (Itest/Vtest), it
can be seen that 70 pM rAgTx2 produced a marked
inhibition (45%) of the K+current of Kv1.1 channels at
all Vtest(Fig 5, 1b) as measured at the end of each 100 ms
test pulse Recombinant PBTx3 produced almost the same
effect by applying 550 nMtoxin on Kv1.2 (Fig 5, 1c) and
500 nMtoxin on Kv1.3 (Fig 5, 1d) channels, whereas the
same degree of inhibition was observed with 70 lMtoxin on
Kv1.1 channels (Fig 5, 1a) This was in close agreement
with the inhibition seen with native PBTx3 (Fig 4A)
The reversal potential for Kv1.1 currents was evaluated
from the kinetics of the tail currents upon repolarization A
tail current/voltage curve (Itail/Vtest) was constructed by
fitting the data with a single Boltzmann distribution
function of the form Itail ¼ Itail,max/{1 + exp[(V1/2–V)/s]}
where Itailis the tail current, Imaxis the maximal tail current,
and s the slope factor of the voltage dependence The peak
amplitudes of the tails were measured at )50 mV and
plotted as a function of the preceding Vtest(Fig 5, 2a,b)
This resulted in a typical fraction open channels/membrane
voltage relationship In the study with rAgTx2 (Fig 5, 2a),
the function in the control situation (n ¼ 4) was
charac-terized by a half-maximal potential (V1/2) and slope (s) of
)19.7 ± 0.7 mV and 10.0 ± 0.7 mV, respectively With
10 pMrAgTx2 (n ¼ 4), V1/2was)20.3 ± 1.3 mV and s
was 10.5 ± 1.3 mV, demonstrating that there was no
significant shift of V1/2 and of the s-value, showing no
effect on the channel gating For the control situation
(n ¼ 4) in the experiment with rPBTx3 (Fig 5, 2b), the V1/
2and s were)19.5 ± 2.7 mV and 10.9 ± 2.7 mV,
respec-tively In the presence of rPBTx3 (n ¼ 4), V1/2and s were
)19.9 ± 1.7 mV and 9.3 ± 1.6 mV, respectively,
suggest-ing that this new toxin did not change the midpoint of the
open channel/voltage curve of Kv1.1 channels Steady-state
Kv1.2 and Kv1.3 currents were converted to conductances
using a reversal potential of)80 mV and fitted to single,
first-order Boltzmann distributions Conductances were
normalized to the maximum estimated from the Boltzmann
fit In control, the function was characterized by a
half-maximal potential (V1/2) of )19.6 ± 3.3 mV and
–22.0 ± 6.0 mV (n ¼ 4) with a slope factor of
7.5 ± 0.3 mV and 9.3 ± 4.7 mV, for Kv1.2 and Kv1.3
channels, respectively With 500 nMrPBTx3, there was no
shift: V1/2, )19.5 ± 1.7 mV and –21.6 ± 4.3 mV and s
9.6 ± 3.3 mV and 8.5 ± 6.3 mV (n ¼ 4) for Kv1.2 and
Kv1.3 channels, respectively (Fig 5, 2c,d)
The induced inhibition by rPBTx3 was
concentration-dependent Fig 6A and B show the dose–response curves of
Kv1 channels to the recombinant toxins The half-maximal
effect on Kv1.2 and Kv1.3 channels was obtained with
547 nMand 492 nM, respectively However, the affinity of rPBTx3 for Kv1.1 channels was very low, with
Kd ¼ 79 lM, showing that rAgTx2 (Kd ¼ 59 pM) has a
1· 106 times higher affinity toward these channels The
Fig 5 (1a–d) The current/voltage (I test /V test ) relationship in control (s) and in the presence (d) of different concentrations of rPBTx3 (a, c, d) on Kv1.1, Kv1.2 and Kv1.3, respectively, and rAgTx2 (B) on Kv1.1 channels expressed in Xenopus oocytes Currents were measured at the end of each 500 ms test pulse In all cases, the effect was reversible (2a, b) Corresponding fraction open channels/membrane voltage curve (I tail /
V test ) relationship, fitted with a Boltzmann function (n ¼ 4) (2a) In the absence of toxin (s), the midpoint (V 1/2 ) and slope factor for Kv1.1 channels were )19.7 ± 0.7 mV and 10.0 ± 0.7 mV, respectively In the presence of rAgTx2 (d), V 1/2 and s were )20.3 ± 1.3 mV and 10.5 ± 1.3 mV (2b) In the control experiment (s) for rPBTx3 on Kv1.1 channels, V 1/2 and s were )19.5 ± 2.7 and 10.9 ± 2.7, respectively, whereas after addition of rPBTx3 (d), they were )19.9 ± 1.7 mV and 9.3 ± 1.6 mV, respectively The residual in maximal fraction open channels induced by application of 10 p M rAgTx2 was 75 ± 1.47% and by application of 70 l M rPBTx3 it was 53.6 ± 9.4% (2c,d) Maximal membrane conductances (G max ) were calculated The steady-state activation curves for the control (s) and in the presence of 500 n M PBTx3 (d) were obtained after fitting with a Boltzmann function I ¼ I c /[1 + exp(–V test –V 1/2 )/s])1 In both cases, for Kv1.2 and Kv1.3, V 1/2 is not shifted by rPBTx3 as illustrated by the dashed lines Slope values (s) for the control and the toxin curves are, respectively, 7.5 and 9.6 for Kv1.2, and 9.3 and 8.5 for Kv1.3 channels.
In all cases, there was no significant shift of V 1/2
Trang 7obtained Kdof rAgTx2 for Kv1.1 was in accordance with
the value reported by Garcia et al [15]
Block was reversible upon washing-out As the toxin
binding was reversible and did not alter channel gating, we
investigated rPBTx3 binding to the channel As has been
explained earlier, blockade is assumed to occur by a simple
bimolecular reaction If the toxin binding to the channel
indeed reflects a bimolecular reaction scheme, the apparent
first-order association rate increases linearly with toxin
concentration and the first-order dissociation rate remains
constant This was indeed the case as shown in Fig 6C,
where the effects of increasing rPBTx3 concentrations on
the kinetics of block on Kv1.2 are illustrated The time
course of activation was fitted using a Hodgkin–Huxley
type model with a 4th power function of the form:
It ¼ A {1–exp[+ (t/s)]4+ C}, with It the macroscopic
and time-dependent current, A the current predicted at
steady-state, s the time constant, and C a constant For a
depolarizing pulse from)90 to 0 mV, the activation kinetics
of Kv1.3 could be fitted with a time constant of
11.2 ± 0.7 ms and 10.94 ± 1.1 ms in the control and in
the presence of 500 nM rPBTx3, respectively (Fig 6D)
Recombinant rPBTx3 did not alter the activation or
inactivation time constants of Kv1.3 channels expressed in
oocytes
Other channels Finally, we investigated the effect of our new toxin on different cloned channels, included in the screening process,
in order to study its selectivity profile Recombinant PBTx3 has no effect on Kir2.1 channels, hERG-type channels, hH1
Na+ channels (plant) KAT channels, cardiac two-pore background K+ channels (cTBAK) and the calcium channel p2X expressed in Xenopus oocytes (data not shown)
D I S C U S S I O N
The number of peptides isolated from distinct phyla, like scorpions [23], sea anemones [24,25], marine cone snails [26] and snakes has increased considerably They have a three-dimensional structure with some conserved motifs [27] but their affinity and specificity towards different targets may vary Those targets include ion channels, present in different tissues In order to increase our knowledge of the structure– function relationship between toxins and ion channels, it is necessary to isolate peptides in scorpion venoms and characterize them as much as possible In this study, we present the purification, primary structure and functional characterization of PBTx3, a novel peptide inhibitor from the venom of the P transvaalicus scorpion PBTx3 was isolated from the venom on the basis of its ability to inhibit the K+ current through cloned voltage-dependent K+ channels (Kv1) expressed in Xenopus oocytes Separation procedures leading to the identification of this novel neurotoxin were performed by gel filtration and reversed-phase HPLC, by using different types of columns, as described previously [28]
The new toxin PBTx3 has a peptidic chain of 37 amino acids and shows similarities with members of the first subfamily of a-K+ scorpion toxins [8], with a fully conserved stretch of residues G25-K26-C27-M28-N29 residing in one of the b sheets, like ChTx The sequence Lys-Cys-XXX-Lys-Cys (X being any amino acid), with the Lys–Cys in antiparallel b sheets and XXX being a tight turn,
is also conserved, as in all other small scorpion toxins that are active on K+channels In order to find structurally significant features in the sequence of PBTx3 (Fig 2B), sequence alignments were performed using the program CLUSTAL1.8 (http://searchlauncher.bcm.tmc.edu:9331/mul-tialign/multialign.html) PBTx3 shows similarities with ChTx (41%) [29], Lqh 15-1 (44%) [30] and ChTx-Lq-2 (38%) [11] from Leiurus quinquestriatus var Hebraeus, BmTx 1 (55%) and 2 (41%) [31] from Buthus martensi Karsch, HgTx 2 (55%) [32] and LbTx (50%) [33] from Centruroides limbatus, IbTx (47%) [34] and TmTx (52%) [35] from Buthus tamulus Alignment of the cysteine residues (C6–C27, C12–C32, C16–C34) showed that it was a novel toxin and that the cysteine motif was highly conserved This cysteine pattern was also found in long-chain scorpion toxins [36] and other defence proteins such as the antibac-terial insect defensin A [37], as well as in plant thionins [38] and potent antifungal plant defensins [39] Disulfide bridges are important in stabilizing the three-dimensional structure
of the toxin, as demonstrated by NMR studies of ChTx [40], iberiotoxin [41] and Lq2 [42] Definitive assignment of the disulfide linkages in PBTx3 is currently unknown but is assumed to mimic that of ChTx and other a-KTx Specific
Fig 6 Dose–response curves of rAgTx2 (A) and rPBTx3 (B) with a K d
value for rAgTx2 of 59 p M (Hill coefficient of 0.9) Each point
repre-sents the mean ± SD from four oocytes The expected K d values for
rPBTx3 on Kv1.1, Kv1.2 and Kv1.3 are, respectively, 79 l M , 547 n M
and 492 n M (Hill coefficients 0.89, 1.41 and 1.16, respectively) (C)
Bimolecular kinetics of PBTx3 interaction Rate constants of blocking
[k on (rPBTx3), d] and dissociation (k off , s) were measured from
volt-age-clamp records as a function of external rPBTx3 concentration.
Each point represents the mean ± SD of three individual
determin-ations (D) Effect of rPBTx3 on activation and inactivation kinetics of
Kv1.3 channels After depolarizing up to 0 mV from a V hold of
)90 mV for 500 ms, the activation and inactivation process in the
presence of rPBTx3 is not changed Both current traces, control and in
the presence of toxin, have been superimposed after scaling of the trace
in presence of rPBTx3.
Trang 8residues in ChTx, responsible for specific properties, are also
present in PBTx3 For example K26 (using PBTx3
numbering), the crucial residue in the interaction with the
pore of voltage-gated K+channels [43], is located in the
centre of the molecule Furthermore G26 (corresponding to
G25 in PBTx3) has been suggested to be important for
appropriate formation of the disulfide pairing [44] and is
also conserved throughout these sequences of all the
members of subfamily 1 of a-KTx, including PBTx3
Because of these similarities and conservation of the
consensus sequence, proposed for a-KTx subfamily 1, this
new toxin is supposed to be the tenth member of the a-K
toxin 1 subfamily Although this novel toxin maintains a
number of expected features, present also in ChTx and
known to be important for the activity, it is unique in some
aspects In contrast with other members of the
ChTx-subfamily, PBTx3 lacks F2 and W14 The latter plays a role
in the interaction of the other members of this subfamily
with residue G380 in the outer vestibule of the Kv1.3
channel [45] The mutation W13L could well be responsible
for the lower affinity of PBTx3 for Kv1 channels, as a
similar decrease in affinity was demonstrated previously for
the W14A mutant of ChTx [45] However, those two
residues are seen only in the toxins known to block BK
channels (large-conductance Ca2+-activated K+channels)
PBTx3 conserves also a higher content of proline residues
(two), but the importance of this is not really clear PBTx3
possesses no N-terminal pyroglutamate, a residue classified
as influential in the functional map of ChTx [46] These
structural differences in PBTx3 together with differences in
the sequence at crucial or influential places (one N-terminal
residue fewer, R22, P23, N24, R30, K33 and P36 versus the
N-terminus, T23, S24, R25, K31, R34 and S37 in ChTx)
may explain why the affinity of rPBTx3 is much lower for
Kv1 channels The toxin is composed of 37 amino-acid
residues, with 11 positively charged groups and three
negatively charged residues, dispersed all over the molecular
surface Groups of strong hydrophobicity (M4, M28, Y35)
and H-binding capacity (S8, S9 N24 and N29) would
suggest that the specific block of the toxin relies upon
hydrophobic as well as polar interactions The
three-dimensional structure of PBTx3 is also related to the
a-KTx1 subfamily Its three-dimensional conformation is
determined by homology modelling (Fig 7) with
hongo-toxin 2 (a-KTx 1.9) as a template for modelling because this
latter toxin shares 55% homology with PBTx3
The a/b scaffold consists of a short a helix (residues
S9–A19) and a b sheet, which is not triple- but
double-stranded in PBTx3 Rather than forming a third b strand as
found for other a-K toxins, the N terminal region of
PBTx3, based in our model, adopts an extended
confor-mation This can be explained by the presence of the
N-terminal end of PBTx3, which is one residue shorter than
that of ChTx It has been shown that toxins acting on SK
channels mostly contain a two-stranded antiparallel b sheet
(leiurotoxin I and PO5), whereas toxins active on Kv
channels mostly have a triple-stranded b sheet Whether
PBTx3 blocks SK channels remains to be investigated The
key feature of ChTx block of the Kv1 channels, a 1 : 1
stochiometry for toxin block of the channel, is also observed
with PBTx3 Although those two toxins could share a
common mechanism for blocking, there are some
quanti-tative differences in the blocking kinetics For instance, the
on rate of rPBTx3 binding to Kv1 channels is 10–100 times slower than that of ChTx for which, depending on the conditions, channels are blocked with an on rate of 0.2–
20· 107
M )1Æs)1 This is not very surprising as ChTx and PBTx3 share only 41% sequence homology Only three positively charged residues are conserved between the two toxins, and two arginine residues and lysine residues are exchanged between the two toxins, located in the a-sheet Of the three residues in ChTx (R25, K27 and R34) crucial to toxin binding and blockade [46], only the K27 is conserved The R34 is mutated to a lysine residue Because of the difference in the length of their side chains, lysine and arginine could have a different effect as also described for other toxins [47] However, structural similarities in this part
of the toxins may underlie the functional similarities observed for the toxins ChTx is a highly basic toxin, with
a net charge of +5 at neutral pH, whereas PBTx3 (still more basic), carries a net charge of +9 (pH range 5–9) For PBTx3, an additional negatively charged D3 is present, and could be an explanation for some of the differences in the association rate constants of the two toxins
Several binding sites of K+channel blocking peptides have been characterized and most of these blockers possess
at least a common diad composed of two functionally important residues, separated by 6.6 ± 1.0 A˚: a positively charged residue and a hydrophobic residue [48,49] Residues
in AgTx2 and ChTx at positions equivalent to Y36 and K27
of PBTx3 have been shown to be critical for channel blocking [50,51] These two residues are also found in anemone K+channel toxins, despite the fact that the three-dimensional folding of scorpion and anemone toxins are quite different [48] Regarding this hypothesis and in correspondence with the diad in ChTx, K26 and the Y35
in PBTx3 are most probably involved in this diad The distance that separates the Ca of the lysine from the centre of the benzene ring of the tyrosine is 6.805 ± 0.406 A˚ We can imagine that the toxin interacts with the channel like a moon lander system and that those two residues play an important role in the interaction with the pore of the channel Our control toxin in the recombinant expression, AgTx2,
Fig 7 A three-dimensional model for PBTx3, constructed by homology modelling The backbone of the molecule is shown in ribbon Residues forming the functional diad (K26 and Y35) are in yellow.
Trang 9represents a very potent blocker of Kv1 type channels.
Mutagenesis studies on AgTx2 identified a set of residues as
functionally important for blocking the Shaker K+channels
(N30, K27, R24, S11, F25, T36, M29 and less important
R31) [52] Three residues are mutated in PBTx3, namely
R27P, F25N and T36Y (AgTx2 numbering) The T36Y
mutation is unlikely to affect drastically the affinity toward
Kv1.3 channels, as it also occurs in other members of the
first group The effect of the R27P and F25N mutations
could be more important, considering the diad hypothesis
Most of the other mutations are located far from the
inter-action surface, upstream from the a helix or within this helix
The sequence of rPBTx3 includes some similarities with
subfamily three, seven and eight of the a-KTx toxins These
toxins all end with a positively charged residue at the
C-terminus, preceded by a proline Functionally, the
recom-binant toxin, lacking the arginine, demonstrates the same
properties as the native toxin (with an additional arginine),
illustrating that this residue is not important for function In
the first b sheet, PBTx3 represents a fully conserved stretch
referred to as the kaliotoxin group: C18-K19-A21-G22
Comparing the S5-P-S6 regions of the three channels, we
can look for specific residues in the pore-forming region that
are different between Kv1.1 channels and Kv1.2 or Kv1.3,
that can possibly explain the selectivity toward these latter
channels The only residue, present in both Kv1.2 and
Kv1.3 and mutated in Kv1.1 is D372 (Kv1.3 numbering)
This residue is probably involved directly in the intimate
interaction with the toxin right at the binding site
Mac-Kinnon et al (1989) observed a substantial reduction on the
binding affinity when the structure of this site was altered by
shortening the side chain (E–D) [13] However, some studies
have shown that the same mutations in highly homologous
K+channels can produce different effects Therefore the
extrapolation of the structural and functional importance of
residues should be done with caution, even with ion
channels belonging to the same family [53]
It is well known that long-chain scorpion neurotoxic
polypeptides from the Buthidae family generally account for
about 10–50% of the crude venom and that short-chain
scorpion peptides appear only in very low quantities in the
venom [54] During the past decade, a number of
approa-ches have been developed to produce toxins For example
PBTx3 is assessed to be only about 0.06% of the venom
Expression of scorpion toxins in Cos-7 cells [55], in insect
cells by means of the Baculovirus system [56], in plants [57],
in NIH/3T3 mouse cells [58] and in yeast [57] led to rather
low yields The first recombinant toxin was described about
10 years ago [59] and different toxins followed We
produced rPBTx3 in order to verify that this peptide was
indeed the inhibitory component in the scorpion venom,
excluding the possibility of the contamination with a peptide
of higher affinity to K+ channels The system chosen to
express PBTx3 in E coli had previously been shown to be
suitable for the production of soluble, correctly folded
spider [60] or scorpion toxins [61] Following the procedures
described in this study, it is feasible to produce 2–4 mg of
homogeneous and biologically active toxin from 1 L E coli
culture The production of fully active rAgTx2 and rPBTx3
requires some in vitro post-translational modifications that
are difficult to control: proteolytic release of the toxin from
the fusion protein and correct forming of the three disulfide
bonds by the six cysteines Based on the chromatographic
profile of a mixture of rAgTx2 and native AgTx2 (AlomoneÒ), which resulted in a single elution peak without additional components, and based on the identical func-tional activity on Kv1, we can assume that the folding process in rAgTx2 was correctly performed In the case of rPBTx3, the elution time of the native and the recombinant toxins were identical and the effect on Kv1 channels was also comparable Therefore, we could also conclude that PBTx3 is not amidated, because peptides of this size with a free or with an amidated residue in the C-terminal position exhibit different retention times on HPLC [62] The reduced peptide could be air oxidized in a concentration-independ-ent manner This was observed previously for other short scorpion toxins acting on Ca2+-activated K+channels (e.g leiurotoxin I and PO5) [63,64] The lack of activity of the fusion proteins is not unexpected as the 44 kDa additional mass could affect significantly the folding and accessibility
of the toxin portion
As mentioned before, just a few studies were performed based on the native venom of P transvaalicus Crude venom of P transvaalicus has been shown to modulate the ChTx binding to aortic sarcolemmal vesicles, in a way that it was able to inhibit ChTx binding in the preparation [34] Inhibitors from scorpions, snakes and bees appear to target primarily either the Shaker-related subfamily of Kv chan-nels or the Ca2+-activated K+channels [15,65,66] In our study, we used a heterologous expression in oocytes of cloned Kv channel proteins To determine which type of voltage-gated K+channel could be sensitive to recombinant PBTx3, electrophysiological experiments were performed
on Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes Kv1.3 channels have been found in several types of cells, in neurons, and in T lymphocytes and have proven to
be highly sensitive to scorpion toxins [67] Analysis of the effects of rPBTx3 on Kv1 channels showed that rPBTx3 mimicked the effects of ChTx ChTx blocks Kv1.2 and Kv1.3 with dissociation constants in the nanomolar range, but does not block Kv1.1, even at 1 lM [68] In parallel, rPBTx3 blocks Kv1.2 and Kv1.3 channels, but with lower channel affinities than those of ChTx The half-maximal blockage of Kv1.2 and Kv1.3 occurred at 547 nM and
592 nM, compared with 6 nM and 1 nM for ChTx [68] Although there is a considerable amount of sequence identity between PBTx3 and other members of subfamily 1
of the a-KTx, the values for the association rates and dissociation rate constants differed from those determined previously for AgTx 2 and ChTx [46,69] We examined the inhibitory effects of rPBTx3 at different membrane voltages Block induced by rPBTx3 was voltage-independent over the range)30 to +20 mV, indicating that this toxin is not very sensitive to the gating state of the channel Channel block by AgTx2 is performed by physical occlusion of the conduction pore [23] The overall channel conductance, measured from the slope of the current–voltage relationship, is not changed
in all cases in the presence of toxin Fig 5 shows activation curves obtained in the absence and presence of extracellular rPBTx3 on Kv1, channels Recombinant PBTx3 does not shift the voltage at which the channels open Also, as demonstrated for Kv1.3, there was no shift in the activation
or inactivation kinetics of those three channels, as demon-strated for Kv1.3 (Fig 6D) For Kv1.1, both the onset and recovery from inhibition were slow Because the toxin does not alter channel Kv1.2 gating and the binding to this
Trang 10channel is reversible, the time constants for relaxation to
equilibrium block upon toxin exposure reflect only the
process of the binding reaction Therefore, the kinetics of
rPBTx3-induced inhibition were consistent with a
bimolec-ular reaction between PBTx3 and Kv1.2 The forward rate
constant for onset of inhibition varied linearly with PBTx3
concentration, while backward rate constant for recovery
from inhibition was independent of PBTx3 concentration
(Fig 6C) The dissociation constants (koff) decreased from
Kv1.1 to Kv1.2 and Kv1.3, in an order that correlates with
the increase of the affinity of rPBTx3 for these channels We
screened a variety of other channels to investigate the
selectivity of rPBTx3, but no modulation was observed
In the future, additional functional characterization
of rPBTx3 on other types of channels is planned where,
for example, Ca2+-activated K+channels and other Kv1
channels (e.g Kv1.6) are good candidates
A C K N O W L E D G E M E N T S
We thank O Pongs for providing the cDNA for the Kv1.2 channel and
C Ulens for the subcloning of the gene encoding the Kv1.2 channel The
Kv1.3 clone was kindly provided by M L Garcia We are grateful to H.
Sentenac to provide the KAT1 clone The hK1 clone was kindly
provided by R G Kallen We also thank E Toth Zsamboki for
providing the P2X clone and Y Kurachi for providing the TBAK clone.
I H and E C are Research Assistants of the Flemish Fund for Scientific
Research (F.W.O.-Vlaanderen) This work was supported by a bilateral
collaboration between Flanders and South Africa (BIL00/36).
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