From the Department of Physiology and Biophysics,
Case Western Reserve University, Cleveland, Ohio 44106, § Molekulare Biologie Neuronaler Signale,
Max-Planck-Institut für experimentelle Medizin,
Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany, and the
Departments of ¶ Biology and
Pathology, University of Utah,
Salt Lake City, Utah 84112
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ABSTRACT |
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-Conotoxin PVIIA
(
-PVIIA), a 27-amino acid toxin from Conus
purpurascens venom that inhibits the Shaker potassium
channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined.
-Conotoxin PVIIA has the following structure.
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Although both -PVIIA and charybdotoxin inhibit the
Shaker channel, they must interact differently. The F425G
Shaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes
-PVIIA
sensitivity.
The precursor sequence of -PVIIA was deduced from a
cDNA clone, revealing a prepropeptide comprising 72 amino acids.
The N-terminal region of the
-PVIIA prepropeptide
exhibits striking homology to the
-, µO-, and
-conotoxins.
Thus, at least four pharmacologically distinct superfamilies of
Conus peptides belong to the same "O" superfamily, with
the
- and
-conotoxins forming one branch, and the
- and
µO-conotoxins forming a second major branch.
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INTRODUCTION |
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The venoms of the 500 species of predatory marine snails belonging
to the genus Conus have proven to be a rich source of
peptidic ligands with high affinity and specificity. The conotoxins are relatively small, disulfide-bridged peptides (12-30 amino acids) that
can readily be chemically synthesized. A large number of peptides that
target voltage-gated calcium and sodium channels, as well as the
acetylcholine receptor, have been characterized. However, the peptides
that target potassium channels in Conus venoms are
relatively unexplored; recently, we described an initial characterization of
-PVIIA,1 a
peptide from the fish-hunting snail Conus purpurascens,
which inhibited the Shaker K+ channel but
had no effects on any voltage-gated Ca2+ or Na+
channel tested (1). The sequence of
-PVIIA bears little
apparent homology to the dendrotoxins or to scorpion and spider toxins, which block the Shaker K+ channels.
The Shaker potassium channel is one of the most intensively investigated neuronal signaling macromolecules. Several polypeptide toxins, notably charybdotoxin and the agitoxins (2), have been used to probe this channel; this approach has made the outer vestibule of the Shaker potassium channel the most well mapped region of any ion channel complex. A novel toxin based on a different structural framework from existing ligands could potentially interact with unmapped extracellular regions of the ion channel complex.
In this report, we describe the successful chemical synthesis of
-PVIIA and determination of the disulfide connectivity
of the biologically active peptide. We have also determined the
predicted precursor sequence for the peptide, derived from the nucleic
acid sequence of a cDNA clone encoding the peptide. These data
confirm the amino acid sequence assignment for
-conotoxin
PVIIA reported previously (1). Finally, we have
investigated interactions between the toxin and chimeric or mutagenized
analogs of the Shaker K+ channel.
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EXPERIMENTAL PROCEDURES |
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Native Peptide Purification--
-Conotoxin PVIIA
was purified from milked venom obtained as described by Hopkins
et al. (3) from specimens of C. purpurascens collected from the Gulf of California. Milked venom was diluted to 10 ml with a solution of 0.1% trifluoroacetic acid, 99.9%
H2O and quickly injected onto a C18 Vydac
preparative column (22.0 × 250 mm, 10-µm particle size, 300-Å
pore size). Elution of peptides was carried out at a flow rate of 20 ml/min, using a gradient of buffer A containing 0.1% trifluoroacetic
acid, 99.9% H2O, and buffer B containing 0.085%
trifluoroacetic acid, 9.915% H2O, 90% CH3CN
as described previously (1). Each peak from the run was collected and
stored at 70 °C as stock solutions, and further purification was
carried out from these stock fractions. A C18 Vydac
analytical column (4.6 × 250 mm, 5-µm particle size) was used
for the secondary purification with flow rate of 1 ml/min and B buffer
containing 0.085% trifluoroacetic acid, 39.915% H2O, 60%
CH3CN.
Partial Reduction and Amino Acid Sequence Analysis--
The
stepwise reduction and differential alkylation of -PVIIA
to determine disulfide connectivity was carried out as described previously (3-5). The alkylated peptides were purified by HPLC, and
sequencing was performed with Edman chemistry on an Applied Biosystems
model 477A Protein Sequencer at the Protein/DNA Core Facility at the
University of Utah; we are grateful to Dr. Robert Schackmann for the
sequence analysis. Mass spectra were measured with a JEOL JMS-HX110
double-focusing spectrometer fitted with a Cs+ gun.
Solid-phase Peptide Synthesis-- The protected peptide resin was built using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as described by Shon et al. (4) with two of the cysteines (position 8 and 20) protected by S-acetoamidomethyl and the other cysteines with S-trityl. Other side chains were protected as follows: pentamethylchromasulfonyl (Arg), t-butyl (Hyp, Asp, and Ser), trityl (Asn, His), and t-butoxycarbonyl (Lys). Initially, a two-step oxidation protocol was used to generate three fully disulfide-bonded peptide isomers as detailed by Monje et al. (6); only one isomer was biologically active. This resulted in a very poor yield and led us to try conditions in which the linear peptide without acetamidomethyl-protected Cys was diluted in 0.1% trifluoroacetic acid to less than 50 µM in the presence of 1 mM reduced and 0.5 mM oxidized glutathione (pH adjusted to 7.5). The equilibrium shift induced by the presence of excess reduced glutathione oxidized the peptide to a number of fully oxidized isomers. The major product, which was >90% of this mixture, proved identical to the native peptide (see "Results") and provided a far better yield than the initial two-step oxidation protocol. Five to six hours at the ambient temperature is usually enough to complete the folding reaction using these conditions.
Identification and Sequencing of C. purpurascens cDNA
Clones--
5 µg of venom duct RNA from C. purpurascens
was annealed to 3 pmol of a poly(T) oligonucleotide, and cDNA was
synthesized by avian myeloblastosis virus reverse transcriptase (5 units; Promega, according to the manufacturer's suggested protocol). The resulting cDNA was used as a template for PCR reaction in 10 10-µl sealed capillary tubes using an Idaho Technology air thermocycler. Each reaction contained 50 ng of template cDNA, 5 pmol each of oligonucleotides corresponding to the 5- and
3
-untranslated regions sequence of
-conotoxin prepropeptides, 5 nmol of each of the four dNPTs, and 0.5 units of Taq
polymerase (Boehringer Mannheim) in a buffer consisting of 50 mM Tris, pH 8.3, 250 µg/ml bovine serum albumin, and 2 mM MgCl2. The PCR consisted of 40 cycles
(94 °C pulse, 54 °C pulse, and 72 °C for 15 s).
Sequencing--
Single-stranded DNA was prepared from putative
-PVIIA clones for sequencing by PCR amplification of 50 ng of plasmid with a pair of vector primers, one of which is
biotinylated, and binding the resulting PCR product to
streptavidin-bound magnetic polystyrene beads (Dynal, Dynabeads M-280
streptavidin). Material for solid-phase sequencing was prepared
according to the manufacturer's suggested protocol, generating
single-stranded nucleic acid, which was sequenced using the Sequenase
version 2.0 DNA sequencing kit, the non-biotinylated vector primer, and
[35S]dATP, according to standard Sequenase protocol.
cRNA Synthesis-- Shaker H4 (7) channel gene was cloned in a Bluescript vector. The Kv1.1 (RCK1; Ref. 8) and the chimeras were in pSGEM. Unique restriction sites, which left the primary sequence unchanged, and point mutations were introduced in Shaker and RCK1 channels by PCR mutagenesis, and the amplified sequences were controlled by sequencing. These silent changes added a XbaI site at the coding sequence for Leu186 (after S1), a NheI site at Ala321 (in front of S5), and a DraIII site at Ser390 (beginning of S6) in RCK1. In Shaker, a NheI site was introduced at the corresponding place (Ala391). The new sites, in combination with the available sites, permitted easy replacement of the corresponding Shaker (S) and RCK1 (R) parts to generate the three chimeras (RRS1-4R, RRS5-6R and SSR5-6S). The pSGEM-plasmid with the Kv1.1 channel and the chimeras were linearized with PacI. The Skaker-bearing plasmid was linearized with HindIII. All cDNA clones were transcribed using T7 RNA polymerase.
Oocyte Expression System-- Oocytes from Xenopus laevis were prepared as described previously (9). mRNA was injected into stage V-VI oocytes. Whole cell currents were recorded 1-5 days after injection under two-electrode voltage clamp control using a Turbo-Tec amplifier (NPI Elektronik, Tamm, Germany) driven by the Pulse+PulseFit software package (HEKA Elektronik, Lambrecht, Germany). The intracellular electrodes were filled with 2 M KCl and had a resistance between 0.6 and 1.0 megohms. Current records were sampled at 4 kHz and low pass-filtered at 1 kHz. The bath solution was normal frog Ringer's containing (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 Hepes, pH 7.2 (NaOH). Leak and capacitive currents were corrected on-line by using a P/n method. Toxin solution was added to the bath chamber by using a Gilson tip pipette. The indicated toxin concentrations correspond to the final concentration in the bath chamber.
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RESULTS |
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Precursor Sequence of -Conotoxin PVIIA--
A
tentative amino acid sequence for
-PVIIA was assigned
previously (1). An analysis of the purified peptide by mass
spectrometry gave a mass (monoisotopic MH+ = 3268.4;
theoretical = 3268.42) consistent with the predicted sequence if
all Cys residues were disulfide-bonded and the C terminus was
not amidated. Both the sequence assignment and the free C terminus were confirmed by chemical synthesis of the peptide (see following section) and by analyzing the sequence of a cDNA clone encoding
-conotoxin PVIIA.
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Chemical Synthesis--
The sequence assignment for
-PVIIA and the free C terminus was confirmed by chemical
synthesis (see "Experimental Procedures"). The synthetic peptide
was biologically active when injected into fish and mice, and proved to
have an elution time identical to that for the native material upon
high performance liquid chromatography (see Fig.
2). The peptide elicited the same
fin-popping syndrome as did native
-PVIIA when injected
into goldfish, and caused the same hyperactivity syndrome seen with
native material when injected intracerebroventricularly into mice.
Thus, the successful chemical synthesis of biologically active
-conotoxin PVIIA validates the sequence assignment of
Terlau et al. (1). The synthesis protocol described
routinely yields 5-10 mg of pure peptide.
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Disulfide Bridging Pattern of -Conotoxin
PVIIA--
The disulfide bridge connectivity of
-PVIIA was analyzed, using the partial reduction
strategy of Gray (10). The spectrum of products obtained after partial
reduction using tris(2-carboxyethyl)phosphine is shown in Fig.
3. The partially reduced intermediates
(labeled PR1 and PR2 in Fig. 3) were further
characterized using the double alkylation strategy described previously
(4). Basically, reduced cysteine residues were alkylated using
iodoacetamide under rapid alkylation conditions, followed by full
reduction and pyridylethylation (using 4-vinylpyridine) of the
remaining cysteine residues. The doubly alkylated products were then
sequenced for pairwise deduction of the disulfide connectivity.
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Electrophysiological Studies--
A comparison of the activity of
-conotoxin PVIIA on the Shaker and rat brain
Kvl.1 K+ channels is shown in Fig.
4. The Shaker K+
channel shows fast inactivation, whereas the Kv1.1 channel exhibits non-inactivating currents. It is evident from this figure that, whereas
the Shaker K+ channel is sensitive to
-conotoxin PVIIA, the cloned Kv1.1 channel from rat
brain is not. To define the region of the channel that has the most
important determinants for toxin binding, complementary chimeras of the
Shaker and Kv1.1 channels were constructed.
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Interaction of -Conotoxin PVIIA with the TEA Binding
Site on the Shaker Channel--
The Shaker potassium
channel is sensitive to external TEA, and mutagenesis has demonstrated
that a specific threonine residue, Thr449, is a major
determinant in the interaction of the channel with TEA (12). Mutation
of the protein at this locus from threonine to tyrosine renders the
Shaker K+ channel more TEA-sensitive (12), and
makes it resistant to a number of channel-blocking toxins including
charybdotoxin (13). Since the experiments on chimeras described above
indicated that
-PVIIA might be interacting as a channel
blocker, we investigated the effects of the T449Y mutation on
sensitivity to
-PVIIA. As shown in Fig.
6, this mutation does in fact render the
channel insensitive to
-PVIIA, consistent with the toxin
acting as a pore blocker directly interacting with the external TEA
binding site. Introducing Lys or Gln in place of Thr449
also resulted in a channel that is insensitive to
-PVIIA
(data not shown).
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-Conotoxin PVIIA and Charybdotoxin Differ in Their
Interaction with the Shaker K+ Channel--
Thus far, all
evidence presented indicates that
-conotoxin PVIIA and
charybdotoxin interact very similarly with the Shaker K+ channel. However, in at least one case, the effect of a
mutation in the Shaker K+ channel has opposite
effects on the two toxins.
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DISCUSSION |
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The purification and preliminary amino acid sequence assignment
for -conotoxin PVIIA has been described by Terlau
et al. (1). In this work, we have rigorously established the
sequence assignment in three ways: 1) the mass spectrometric analysis
yielded a value consistent with the previous sequence assignment; 2) a cDNA encoding
-conotoxin PVIIA was identified and
sequenced revealing a precursor consistent with the expected amino acid
sequence of the mature toxin; and 3) biologically active peptide
identical to the natural material was chemically synthesized and
folded. The synthetic, oxidized peptide was shown to co-elute with the native material on HPLC. In addition, the three disulfide linkages have
been assigned. All the data are consistent with the conclusion that
-conotoxin PVIIA shares a common disulfide bridging
pattern with the
- and
-conotoxins.
The general biochemical features of -conotoxin PVIIA are
similar to the
-conotoxin family of peptides, which block certain subtypes of voltage-gated calcium channels.
-Conotoxin
PVIIA has the same disulfide pattern as
-conotoxins and
similarly sized intervals between cysteine residues. Furthermore, like
the
-conotoxins,
-PVIIA has a large number of
positive charges. However, compared with
-conotoxins as a group,
-conotoxin PVIIA has a greater preponderance of
hydrophobic residues, as well as a greater number of negative charges
leading to a smaller net positive charge.
The prepropeptide sequence of the -conotoxin PVIIA
precursor establishes that it belongs to the same superfamily of
Conus peptides as do the
-conotoxins, the
-conotoxins,
and the µO-conotoxins; this superfamily of peptides has previously
been referred to as the O superfamily. Apart from the shared disulfide
bonding pattern, homology within this Conus peptide
superfamily may not be obvious when the individual mature
peptide sequences are compared. However, Table
I demonstrates that all members of the
superfamily share conserved regions in their prepropeptide sequences,
particularly in the signal sequence region. A comparison of precursor
sequences indicates that the
-conotoxins are significantly more
closely related to the
-conotoxins than to the
- and
µO-conotoxins. Thus, there may be two main branches of the O
superfamily: peptides targeting K+ and Ca2+
channels (i.e. the
- and
-conotoxins) and peptides
that target Na+ channels (i.e. the
- and
µO-conotoxins).
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The O superfamily of peptides has diverged to the point where
individual peptides target all the major classes of voltage-gated ion
channels. We note that the -conotoxin PVIA precursor,
from the same fish-hunting snail as
-conotoxin PVIIA, is
more closely related to
- and µO-conotoxin precursors from
snail-hunting cones than to
-PVIIA found in the same
venom. This suggests that the divergence of the two branches of the O
superfamily predates the divergence of fish-hunting and snail-hunting
cone snails. Although
-conotoxin PVIIA is the first
potassium channel-targeted toxin so far described from Conus
venoms, preliminary results indicate that peptides targeted to
potassium channels are likely to be widely distributed among different
Conus venoms.2
-Conotoxin PVIIA should be a useful probe for the
Shaker potassium channel. We have defined a region that
contains the major determinants for
-conotoxin PVIIA
binding in the Shaker potassium channel complex. For both
charybdotoxin and
-conotoxin PVIIA, the interaction
sites that confer high affinity binding are within the S5-S6 loop (for
-PVIIA, see results on chimeras above; for charybdotoxin, see Ref. 2). However, although the two toxins interact
with the same general region, there are clearly different microsite
interactions. Thus, when Phe425 of Shaker is
mutated to a glycine, the KD for block by
charybdotoxin increases by over 1000-fold, from 120 nM to
about 70 pM (16). In contrast, the same mutation resulted
in a channel insensitive to 1 µM
-conotoxin
PVIIA (see Fig. 6). The effects of the F425G mutation on
charybdotoxin were interpreted to be due to a reduction in steric
hindrance; the loss of
-PVIIA sensitivity by the mutant
channel clearly indicates strikingly different interactions with this
locus by the two toxins.
The two toxins are similar in their interaction with a residue known to
be a strong determinant of external TEA binding, threonine 449 (12).
The mutation T449Y results in a channel insensitive to 1 µM -PVIIA. Introducing lysine at the same
position (T449K) also results in a channel insensitive to
-PVIIA (data not shown). Thus, although the two toxins
clearly do not interact with the channel target site in the same way,
they may both block functionally important loci for channel function
such as the TEA binding site.
The remarkable amount of work that has been done using charybdotoxin as
a probe for the Shaker channel serves as a useful paradigm
for defining the -conotoxin:Shaker K+ channel
complex, as well as for evaluating structural proposals of the outer
channel vestibule based on scorpion toxin data. Clearly, an essential
requirement before such structure-function work can proceed
productively is a determination either by multidimensional NMR
techniques or by x-ray crystallography of the three-dimensional structure of
-conotoxin PVIIA. Such a structural
analysis has been
initiated.3
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ACKNOWLEDGEMENTS |
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We thank M. Kuhnert for help in generating the chimeras, S. Voigt for excellent technical assistance, C. Miller for the clone of Shaker H4, and M. Hollmann for the pSGEM vector.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant PO1 GM48677 (to B. M. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom all correspondence should be addressed: University of Utah, 201 S. Biology, Salt Lake City, UT 84112. Tel.: 801-581-8370; Fax: 801-585-5010.
1
The abbreviations used are:
-PVIIA,
-conotoxin PVIIA; HPLC, high
performance liquid chromatography; PCR, polymerase chain reaction; TEA,
tetraethyl-ammonium.
2 H. Terlau, R. Jacobsen, and B. M. Olivera, unpublished results.
3 K.-J. Shon, unpublished results.
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REFERENCES |
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