From the AG Molekulare und Zelluläre
Neuropharmakologie, Max-Planck-Institut für Experimentelle
Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany, the
§ Department of Animal Physiology, University of
Osnabrück, Barbarastr. 11, 40069 Osnabrück, Germany, and
the ¶ Department of Biology, University of Utah,
Salt Lake City, Utah 84112
Received for publication, June 14, 2002, and in revised form, October 16, 2002
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ABSTRACT |
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Voltage-gated ion channels determine the membrane
excitability of cells. Although many Conus peptides that
interact with voltage-gated Na+ and Ca2+
channels have been characterized, relatively few have been identified that interact with K+ channels. We describe a novel
Conus peptide that interacts with the Shaker
K+ channel, Basic research on voltage-gated ion channels advances on two broad
fronts: first, the identification and characterization of the numerous
molecular isoforms that comprise each voltage-gated ion channel family.
A different stream of research focuses on a few model systems
intensively to uncover basic mechanistic insights. For both of these
contrasting facets of ion channel research, the small peptides made by
predatory cone snails (conotoxins) (1, 2) have considerable potential.
Thus, the The first Conus peptide shown to target a voltage-gated ion
channel was µ-conotoxin GIIIA, which was discovered and characterized two decades ago (4-8). A characteristic feature of all µ-conotoxins is the arrangement of disulfide cross-links in the primary
sequence, the µ-conotoxin pattern can be recognized by the following
pattern of Cys residues: -CC In this report, a peptide from Conus radiatus venom ducts
that has the same class III scaffold as the µ- and the Synthesis--
The peptides were synthesized on Rink amide resin
using Fmoc (N-(9-fluorenyl)methoxycarboxyl) chemistry and
standard side chain protection except for the cysteine residues. For
all the peptides, the cysteine side chains were trityl protected.
Peptides were removed from the resin as described previously (11, 12).
Preparative purification of the linear peptides was carried out by high
performance liquid chromatography with either a 5-55 or
10-50% gradient of 0.1% trifluoroacetic acid and 0.1%
trifluoroacetic acid, 60% acetonitrile (buffer B60). The standard
one-step oxidation protocol (13) was used to fold the peptides. Fully
oxidized peptides were purified by preparative high performance liquid
chromatography using either a 5-55 or 10-50% gradient of B60.
Electrophysiological Methods--
The Xenopus
expression system was used for investigating the potential effects of
The IC50 values for the block of Shaker
wild-type and the Shaker K427D channels were calculated from
the peak currents at a test potential of 0 mV according to
IC50 = fc/(1-fc) × [Tx], where fc is the fractional
current and [Tx] is the toxin concentration. For the binding of
Open Channel Binding--
From the ratio of the currents under
control and toxin conditions a single exponential relaxation of the
block is observed (see Fig. 5) that can be interpreted by a simple
bimolecular reaction scheme,
Closed Channel Binding Molecular Biology--
cDNA sequences encoding Cloning and Synthesis of the
The predicted mature peptide that was chemically synthesized is shown
in the bottom of Table I. Procedures used in the chemical synthesis and
folding of the peptide are detailed under "Experimental Procedures."
Biological Activity and Electrophysiological
Characterization--
The peptide elicited obvious symptomatology upon
injection into mice both intracerbrovascular and intrathecal. When 4 nmol of the synthetic peptide were injected by the intracerbrovascular route, seizures were observed. However, when the peptide was injected intraperitoneal into mice, there were no visible effects.
Electrophysiological experiments using amphibian nerve-muscle
preparations were similarly unaffected by 10 µM of the peptide.
Because the peptide has a Class III framework similar to the
µ-conotoxins (which are sodium channel ligands), the effects of the
synthetic
The peptide has been tested on nine different cloned potassium
channels. No activity (with 10 µM peptide) was detected
on Kv1.1, Kv1.3, Kv1.4, Kv2.1, Kv3.4, Kv4.2, herg, and r-eag
K+ channel clones expressed in oocytes. However, when the
peptide was tested on the Shaker K+ channel, an
inhibition of channel conductance was observed as shown in Fig.
2. The inhibition is readily reversible
as shown in the bottom panel of Fig. 2A. The
IC50 for the Shaker channel obtained from
measuring the peak currents is 1.21 ± 0.25 µM
(n = 5 dose-response experiments, see Fig.
2B). The Hill coefficient is ~1, suggesting that binding
of a single toxin molecule is sufficient to inhibit the
Shaker channel.
Studies Using Shaker K+ Channel Mutants with Single
Residue Substitutions--
The interaction of C. radiatus
peptide with a number of mutants of the Shaker potassium
channel were assessed. Many ligands that decrease the conductance of
the Shaker K+ channel bind to the outer
vestibule of the Shaker channel. Among the key amino acids
in this general region found to affect the affinity of other
Shaker K+ channel ligands are
Phe425, Lys427, and Thr449. We
therefore determined whether single amino acid substitutions at these
loci might affect the affinity of the C. radiatus peptide for the Shaker channel.
The results for three different amino acid substitutions (F425G, K427D,
and T449Y) are shown in Fig. 3. The
different substitutions show strikingly different effects; two of the
mutant channels, F425G and T449Y (the latter affects the tetraethyl
ammonium sensitivity of the Shaker channel), were
found to be much more resistant to the C. radiatus peptide.
In contrast, the K427D mutant exhibited about a 10-fold greater
affinity for the toxin (IC50 = 109 ± 61 nM, n = 5) than was observed for the
wild-type Shaker channel. A smaller increase is observed
when Lys427 is substituted with a neutral amino acid
leading to an IC50 of 180 ± 27 nM
(n = 3) for the K427N substitution. The results in Fig.
3 reveal that substitution of any of the three residues, believed to be
near the extracellular opening of the channel pore, significantly
affects toxin affinity. Thus, the three AAs appear to be significant
determinants for the peptide to bind to the Shaker
K+ channel, with either increases or decreases in affinity
observed.
The data are consistent with the C. radiatus peptide
blocking the conductance of the Shaker K+
channel by interactions with the outer vestibule region. Presumably, toxin binding would block Shaker channel conductance by
impeding transit of K+ through the extracellular opening of
the pore.
Effects on a Fish K+ Channel: The Sha1 K+
Channel from Trout--
Because C. radiatus is believed to
be a fish-hunting cone snail, the results above suggest that the
presumptive physiologically relevant molecular target is a
voltage-gated K+ channel in fish. We tested one teleost
K+ channel available as a cDNA clone, the Sha1 channel
from trout (20). We chose the Sha1 channel because the results with
Shaker showed that the K427D Shaker mutant has a
higher affinity than wild-type, and in the trout sequence, the
homologous position to Lys427 in Shaker has a
Glu residue (see Table II).
The results are shown in Fig.
4. The Sha1 channel, which is a
noninactivating voltage-gated K+ channel, is more potently
inhibited by the C. radiatus peptide than is the
Shaker channel. At a concentration of 1 µM
State Dependence of Shaker K+ Channel
Inhibition--
We evaluated whether the affinity of the peptide
changed as a function of the state of the Shaker channel.
The open channel properties were investigated by relaxation of partial
block during step depolarizations. For this work,
Double-pulse protocols were used to characterize the
reequilibration of closed channel binding (see Terlau et
al., Ref. 16). The data derived from the two types of
experiments were used to calculate kinetic parameters
(Kon, Koff, and
IC50) for both the open and the closed states of the
Shaker-
For all three channel types the affinity of the toxin for the open
state measured at 0 mV is about three to four times lower compared with
the closed state. The results show that the affinity of
Furthermore, Table III shows that the major reason for the lower
affinities of the
The data shown in Table III indicate that the negatively charged
residues in Shaker- The peptide characterized above, although structurally related to
the µ-conotoxins that block voltage-gated Na+ channel
conductance, clearly inhibits the Shaker K+
channel. We designate this peptide as The characterization of It was unexpected that a scaffold well known for sodium
channel-targeted ligands would also be used by a Conus
species to target potassium channels. In retrospect, because these ion
channels belong to the same superfamily, the observation can be
rationalized ex post-facto. As will be detailed elsewhere, the
µ-conotoxins and The sequences of two µ-conotoxins and a M-conotoxin RIIIK from Conus
radiatus. The peptide was chemically synthesized. Although
M-conotoxin RIIIK is structurally similar to the µ-conotoxins that
are sodium channel blockers, it does not affect any of the sodium
channels tested, but blocks Shaker K+ channels.
Studies using Shaker K+ channel mutants with
single residue substitutions reveal that the peptide interacts with the
pore region of the channel. Introduction of a negative charge at
residue 427 (K427D) greatly increases the affinity of the toxin,
whereas the substitutions at two other residues, Phe425 and
Thr449, drastically reduced toxin affinity. Based on
the Shaker results, a teleost homolog of the
Shaker K+ channel, TSha1 was identified as a
M-conotoxin RIIIK target. Binding of
M-conotoxin RIIIK is
state-dependent, with an IC50 of 20 nM for the closed state and 60 nM at 0 mV for
the open state of TSha1 channels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxins, one family of conotoxins, have become
standard reagents for discriminating among Ca2+ channel
subtypes (3), and characterizing the functional role of each subtype.
The µ-conotoxins that block voltage-gated channels are used as probes
for the outer vestibule of the channel pore. The subject of this report
is a novel conotoxin that has promising properties to be an important
reagent for structure/function studies of the Shaker
K+ channel, arguably the most intensively studied of all
voltage-gated ion channels. However, the characterization of the
peptide also defines a new family of conotoxins, the
M family, that
should provide a set of new ligands specific for different K+
channel isoforms.
C
C
CC-, now defined as a class III
(or M-1) conotoxin scaffold (9). After the discovery of the
µ-conotoxins, other groups of Conus peptides with three
disulfide bonds (the
-conotoxins,
-conotoxins, µO-conotoxins,
and the spasmodic peptides) were characterized, but these had a
different arrangement of Cys residues. Only one other family of
conopeptides with a class III disulfide framework has been
characterized, the
-conotoxins (10). The latter are noncompetitive
antagonists of nicotinic acetylcholine receptors.
-conotoxins is characterized. Despite its structural affinities, the C. radiatus peptide proved to have an entirely different
pharmacological specificity: it affects the Shaker
K+ channel and is therefore the defining member of a new
family of Conus peptides, the
M-conotoxins. We also
identify a putative K+ channel target in teleost fish, the
presumed prey of C. radiatus. The results demonstrate that
the class III framework first elucidated in the µ-conotoxins has also
been exploited by the cone snails in the course of their evolution to
target a diversity of voltage-gated and ligand-gated ion channels.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M-conotoxin RIIIK on voltage-gated Na+ and
K+ channels. Oocytes from Xenopus
laevis were prepared as described previously (14, 15). Frogs
were anesthetized with 0.2%
Tricaine1 in ice water for
surgery. Following cRNA injection, the oocytes were incubated 1-5 days
to allow expression of the protein. Prior to the electrophysiological
measurements, the vitelline membranes of the oocytes were removed
mechanically with fine forceps. cRNAs encoding various cloned
Na+ and K+ channels to be tested were prepared
by standard techniques. Whole cell currents were recorded under
two-electrode voltage-clamp control using a Turbo-Tec amplifier (NPI
Electronic, Tamm, Germany). The intracellular electrodes were filled
with 2 M KCl and had a resistance between 0.6 and 1 megaohm. Current records were low-pass filtered at 1 kHz
(K+ channels) or 3 kHz (Na+ channels) (
3dB)
and sampled at 4 or 10 kHz, respectively. Leak and capacitive currents
were corrected online by using a P/n method. 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). Lyophilized
M-conotoxin RIIIK was dissolved in normal frog Ringer's, diluted to
the final concentration, and added to the bath chamber. All electrophysiological experiments were performed at room temperature (19-22 °C).
-conotoxin PVIIA to Shaker channels, it was shown that
this is an approximation to obtain the affinity of the toxin to the
closed state of the channel (see below). Data are given as mean ± S.D. The kinetic parameters of the state-dependent block for the different channels investigated were obtained as described in Ref. 16.
where {U} represents the toxin-free channels and
{B} the channels bound to a toxin molecule. From the
experimental parameters
and U it is possible to evaluate
KO, koffO,
konO according to the following
inverse relationships:
The parameters of toxin
binding to the closed state were obtained by performing a similar
analysis for the currents obtained from double pulse protocols
(16).
M-RIIIK
were determined from a directionally cloned cDNA library prepared
from poly(A)+ RNA isolated from C. radiatus
venom as described previously (17, 18). Total DNA from the library was
isolated by standard methods (19), and the DNA using the polymerase
chain reaction in an Air Thermo-Cycler (Idaho Technology, Salt
Lake City, UT); the reaction mixture (10 µl) contained 50 ng of total
DNA, 0.5 µM oligonucleotide primers corresponding to
conserved nucleotide sequences at the 5'- and 3'-untranslated regions
of the µ-conotoxin gene, 50 mM Tris-HCl, pH 8.5 (25 °C), 2 mM MgCl2, 250 µg/ml bovine serum albumin, 0.5 mM of each dNTP, and 0.5 units of
Taq polymerase. PCR was carried out in capillary tubes with
the thermocycler set at a denaturation temperature of 94 °C for
0 s, annealing at 54 °C for 0 s, and elongation at
72 °C for 15 s for 30 cycles. The PCR products were cloned and
sequenced using standards methods. The predicted toxin sequences were
compared with other known members of this family. The peptide encoded
by one of the cDNAs, which was subsequently designated
M-RIIIK,
was synthesized and characterized using electrophysiological assays
(see section above). The wild-type,
6-46, and substitution mutant
clones of the Shaker K+ channel were a generous
gift of Dr. Martin Stocker.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M-conotoxin--
A cDNA clone
from a C. radiatus venom duct library was sequenced and the
predicted mature peptide sequence deduced from rules now standard for
conopeptide precursors. The predicted sequence, one including the
expected post-translational processing of the original ribosomally
translated polypeptide, is shown in Table I. Because in all known µ-conotoxins
and
-conotoxins (the groups with amino acid sequences most related
to that of the mature
M peptide), proline residues are always found
to be hydroxylated, the peptide is inferred to have a hydroxyproline
residue at all loci encoded by a proline codon. The sequence N-terminal
to the mature toxin contains a canonical dibasic signal for proteolytic cleavage (underlined in Table I), whereas the C-terminal amino acid
sequence predicted by the clone would be expected to be
post-translationally processed to an amidated C-terminal threonine
residue.
Nucleic acid sequence of cDNA clone and predicted processing of
peptide
M peptide on three cloned Na+ channel subtypes
(i.e. Nav1.2 (rat brain type II),
Nav1.4 (rat skeletal muscle), and Nav1.5 (mouse
cardiac channel) subtypes) expressed in Xenopus oocyte were
examined (see "Experimental Procedures"). At a concentration of 2 or 10 µM, the peptide did not show any detectable effect
on the currents produced by these cloned sodium channel subtypes. This
is shown in Fig. 1 for
Nav1.4, which is a high affinity target of µ-conotoxins
GIIIA and PIIIA.
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Fig. 1.
M-conotoxin RIIIK does not
block Nav1.4-mediated currents. Upper
panel, whole cell currents recorded from an oocyte expressing
Nav1.4 Na+ channels evoked by test potentials
from
80 to +60 mV in steps of 10 mV. Addition of 10 µM
M-conotoxin RIIIK results in no effect on the evoked currents
(middle panel), which is also demonstrated by the
current-voltage relationships (lower panel). The
dashed line corresponds to zero current.
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Fig. 2.
M-conotoxin RIIIK blocks
Shaker-mediated currents. A, whole
cell currents recorded from an oocyte expressing Shaker
K+ channels evoked by test potentials to 0, 20, and 40 mV
(upper panel). Addition of 2 µM
M-conotoxin
RIIIK results in a block of the currents (middle panel),
which is reversible (lower panel). The dashed
line corresponds to zero current. B, dose-response
curve for the block by
M-conotoxin at a test potential of 0 mV
(n = 5).
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Fig. 3.
Mutations in the pore do affect the binding
of M-conotoxin RIIIK to Shaker
channels. Upper panel, whole cell currents from
oocytes expressing the mutated channel of Shaker H4.
Mutating the phenylalanine to glycine (F425G) results in a
channel that is insensitive to 2 µM
M-conotoxin RIIIK
(left panel). Mutating the lysine 427 to an aspartate
(K427D) results in a channel with increased sensitivity to this toxin
(middle panel). Mutating the threonine at position 449 to
tyrosine (T449Y) results in a channel that is insensitive to 2 µM
M-conotoxin RIIIK. Voltage steps are as described
in the legend to Fig. 2.
Comparison of Shaker and TSha1 K+ channel sequences
M-conotoxin RIIIK the evoked currents are almost completely
inhibited. The inhibition is reversible, as well as
voltage-dependent. The effects of the toxin as a function
of test potential are shown in the bottom panel of Fig. 4.
These results directly establish that the toxin is able to block the
conductance of a vertebrate voltage-gated K+ channel.
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Fig. 4.
The trout homolog of Shaker
channels, TSha1, is blocked by M-conotoxin RIIIK.
A, whole cell currents recorded from an oocyte expressing
TSha1 K+ channels evoked by test potentials to 0, 20, and
40 mV (upper panel). Addition of 1 µM
M-conotoxin RIIIK almost completely blocks the currents
(middle panel) in a reversible manner (lower
panel). The dashed line corresponds to zero current.
B, IV-relationship of the evoked current in the absence and
presence of 1 µM
M-conotoxin RIIIK.
6-46 channels of
Shaker lacking the N-type inactivation were used: this made
it easier to evaluate unblocking of open channels. A sample of results
obtained are shown in Fig. 5; the unblock
follows an exponential time course and is
voltage-dependent. Similar results were obtained for
Shaker-
6-46 K427D and the TSha1 (Fig. 5).
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Fig. 5.
The block of M-conotoxin RIIIK of
Shaker-
6-46, Shaker-
6-46 K427D, and
TSha1 is state-dependent. A, whole cell currents
recorded from oocytes expressing Shaker-
6-46,
Shaker--
6-46 K427D, or TSha1 K+ channels
evoked by test potentials to 0, 20, and 40 mV under control conditions
(upper panel) and after addition of the indicated amounts of
M-conotoxin RIIIK (lower panel). B, current
ratios obtained for the three test potentials showing a single
exponential relaxation of the probability of the channels to be
unblocked obtained by calculating
IToxin/IControl.
6-46 channel, Shaker-
6-46 K427D,
and the TSha1 from trout (see Table III).
The calculations demonstrate that binding of this toxin to open
versus closed channels is very different, i.e.
that the toxin interactions with the Shaker channel are
state-dependent.
Summary of KD, kon, and koff values
M-conotoxin
RIIIK to the open and closed state of the different channels are
calculated as described under "Experimental Procedures" (see also
Ref. 16).
M-conotoxin
RIIIK to Tsha1 from trout is about 20 nM for the closed and
60 nM for the open state measured at 0 mV. This
demonstrates that Tsha1 is a high affinity target for this peptide. The
other data are in accordance with the results obtained with
Shaker wild type and K427D channels (with inactivation;
Figs. 2 and 3) demonstrating that for fast inactivating channels the
calculation of the IC50 from the peak currents is an
approximation for the affinity to the closed state of those channels
(see Terlau et al., Ref. 16).
M-peptide to the open state of the channels is an
increase in koff(O) but also the
kon(O) is affected. The comparison
of the kinetic parameters of the binding for the three different
channel reveals that the koff(O) for
open channel binding for Tsha1 is about 20 times smaller than in the
Shaker--
6-46 channel. In contrast, the
kon(O) in Tsha1 is only three times
higher than in the Shaker--
6-46 channel. The binding
kinetics of
M-RIIIK to the closed state of the three channel types
investigated is slower than the binding to the open state.
6-46 K427D and the TSha1 channel
strongly affect kon(C), which is
almost identical in these two channels, but about four times higher
than in the Shaker-
6-46 channel. In contrast, the koff(C) differs by a factor of three
between Shaker-
6-46 and Shaker-
6-46 K427D
channels, but by a factor of 10 between the Shaker-
6-46 K427D channel and Tsha1. This indicates that a negatively charged amino
acid at residue 427 is a key determinant for rapid association, whereas
koff(C) is primarily influenced by
other residues.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M-conotoxin RIIIK, the first
member of a new family of conotoxins targeted to K+ channels.
M-RIIIK is noteworthy in several respects.
First, the discovery that
M-RIIIK targets the Shaker potassium channel provides a new scaffold for a Shaker
channel ligand, the smallest disulfide cross-linked framework so far
characterized for any polypeptide antagonist of K+
channels. Because this is the most intensively studied voltage-gated ion channel both from a molecular and functional perspective, the
availability of a novel framework in a small polypeptide ligand provides a new platform for examining the topology of this and related
channels. We have also established that the interaction of
M-conotoxin RIIIK with the Shaker channel is
state-dependent. Thus, the peptide is a potentially useful
probe for conformational changes that occur in the transition of a
voltage-gated ion channel from a closed to an open state.
M-conotoxin RIIIK belong to the same gene
superfamily of conopeptides.
-conotoxin are compared
with the
M-conotoxin in Table IV. The
M-conotoxin is distinctive in having a longer first loop, between
the second and third cysteine residues. Structure/function studies on
the µ-conotoxins have established that the arginine residue indicated
by the arrow (Arg13 in µ-conotoxin GIIIA) is a
critical residue for blocking voltage-gated sodium channels, because
the charged guanidino group of the arginine residue is believed to
functionally block the pore. In contrast, both the
-conotoxins and
M-conotoxin RIIIK lack this critical arginine residue. These
differential biochemical characteristics provide a guide in the search
for, and identification of, additional members of the
M-conotoxin
family. Considerable work has been done on µ-conotoxin/sodium
channel interactions. Whether the orientation of
M-conotoxin within
the potassium channel is analogous to the orientation of µ-conotoxins
in the outer vestibule region of sodium channels remains to be
determined.
Comparison of M-conotoxin RIIIK to other M-superfamily Peptides
and
-PVIIA
M-RIIIK. Note that the
M-, µ-, and
-conotoxins all
have the same pattern of Cys residues, whereas
-PVIIA, the only
other peptide that also blocks K+ channels, has an entirely
different arrangement of Cys.
M-Conotoxin RIIIK is not the only conopeptide known to inhibit the
Shaker K+ channel by binding to the outer
vestibule. This was first demonstrated for
-conotoxin PVIIA from
Conus purpurascens (21-23). Although both C. radiatus (the source of
M-RIIIK) and C. purpurascens are probably fish-hunting, they are not closely related species as
judged by available molecular phylogeny data for the genus Conus (24, 25). Because of the accelerated evolution of
venom peptides during speciation through focal mutation (1, 2, 26-28),
the different groups of Conus species use a different
spectrum of conotoxin families as major ligands in their venoms. The
results presented here establish that two different species of cone
snails have evolved structurally and genetically unrelated peptides, both of which block the Shaker K+ channel. The
two peptides have entirely different structural scaffolds;
M-RIIIK
is most closely related to the µ-conotoxins, whereas
-PVIIA has
the greatest structural similarity to the
-conotoxins, which target
voltage-gated Ca2+ channels. Even more conopeptides have
been found that target the Shaker K+
channel,2 and which are
genetically and structurally unrelated to either PVIIA or
M-RIIIK.
Thus, screening a broad range of Conus venoms makes it
possible to identify a structurally diverse set of ligands that target
a given ion channel subtype.
The effect of Shaker K+ channel amino acid
substitutions on the interaction with M-conotoxin RIIIK has provided
insight into the physiologically relevant channel target of the
peptide. The discovery that the K427D Shaker mutant had a
higher affinity for the toxin suggested that the physiological target
of this peptide might have a negatively charged residue at the
homologous locus. Because C. radiatus is believed to be a
piscivorous Conus species, we examined the sequences of
recently cloned teleost channels (20) related to the Shaker
potassium channel. Because the Sha1 channel from trout had a negative
residue at this position, we tested the peptide on the trout channel
expressed in oocytes. The trout Sha1 channel was potently inhibited by
M-conotoxin RIIIK with an IC50 of 20 nM for
the closed state. Thus, this teleost voltage-gated K+
channel subtype is a better target for
M-conotoxin RIIIK than Shaker, exhibiting an almost 50-fold higher affinity.
Although little is known about the true teleost prey of Conus
radiatus, we postulate that the actual high affinity target of
M-conotoxin RIIIK is a voltage-gated K+ channel related
to the trout Sha1 channel. However, the full spectrum of K+
channels in teleost fish has not yet been elucidated.
Previously, it has been suggested that positively charged toxins have an accelerated dissociation from open channels because of the voltage-dependent occupancy of a site at the outer end of the conducting pore by a K+ ion (16). The site has been postulated to also be occupied by external cations in closed channels, thereby antagonizing the association rate. Our results with the C. radiatus peptide, which is positively charged, are generally consistent with this model and suggest that electrostatic interactions between the peptide and K+ ions in the pore are a major factor in state-dependent binding.
Finally, we speculate on the endogenous biological role of
M-conotoxin RIIIK. One potential function for K+
channel-targeted Conus peptides was previously proposed:
that they serve as components of the "lightning-strike cabal" of
toxins that cause excitotoxic shock, a physiological strategy to
instantaneously immobilize prey (2, 21). Thus, a reasonable hypothesis
is that
M-conotoxin RIIIK targets a K+ channel subtype in
peripheral axons similar to the trout Sha1 channel, and in combination
with other excitatory peptides (such as the
-conotoxins that inhibit
Na channel inactivation), causes a massive depolarization of peripheral
axons near the venom injection site. This elicits trains of action
potentials propagated bidirectionally, in effect, the action potentials
directed centrally allow the toxins to functionally bridge the
blood-brain barrier; the end point is equivalent to a tonic/clonic
seizure, resulting in a very rapid tetanic paralysis of the fish prey.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Martin Stocker for the Shaker mutant clones, and Drs. Doju Yoshikami and John Wagstaff for some of the in vivo and electrophysiological assays. We thank Drs. Klaus Benndorf and Thomas Zimmer for Nav1.5 cDNA, Dr. Mark Keating for herg cDNA, and Dr. Jane Dixon for r-erg cDNA.
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FOOTNOTES |
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* This work was supported by Biofuture Prize Förderkennzeichen 0311859 from the German Ministry of Education and Research (to H. T.) and National Institutes of Health Grant GM 48677 (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 correspondence should be addressed: Dept. of Biology,
University of Utah, 257 South 1400 East, Salt Lake City, UT 84112. Tel.: 801-581-8370; Fax: 801-585-5010; E-mail:
olivera@biology.utah.edu.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M205953200
2 J. Imperial, H. Terlau, and B. Olivera, unpublished data.
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ABBREVIATIONS |
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The abbreviation used is: Tricaine, 3-aminobenzoic acid ethyl ester.
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REFERENCES |
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19. | Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, , Third Edition , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
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22. |
Shon, K.,
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23. |
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