(Received for publication, August 15, 1994; and in revised form, November 4, 1994)
From the
We describe a new peptide conotoxin affecting sodium current
inactivation, that competes on binding with -conotoxin TxVIA
(
TxVIA). The amino acid sequence of the new toxin, designated
conotoxin NgVIA (NgVIA), is SKCFSOGTFCGIKOGLCCSVRCFSLFCISFE (where O is trans-4-hydroxyproline). The primary structure of NgVIA has an
identical cysteine framework and similar hydrophobicity as
TxVIA
but differs in its net charge. NgVIA competes with
TxVIA on
binding to rat brain synaptosomes and molluscan central nervous system
and strongly inhibits sodium current inactivation in snail neurons, as
does
TxVIA. In contrast to
TxVIA, NgVIA is a potent paralytic
toxin in vertebrate systems, its binding appears to be
voltage-dependent, and it synergically increases veratridine-induced
sodium influx to rat brain synaptosomes.
TxVIA acts as a partial
antagonist to NgVIA in rat brain in vivo. NgVIA appears to act
via a receptor site distinct from that of
TxVIA but similar to
that of Conus striatus toxin. This new toxin provides a lead
for structure-function relationship studies in the
-conotoxins and
will enable analysis of the functional significance of this complex of
receptor sites in gating mechanisms of sodium channels.
Voltage-dependent sodium channels are integral plasma membrane
proteins responsible for the rapidly rising phase of action potentials
in most excitable tissues and as such are specifically targeted by many
neurotoxins. These toxins occupy at least six identified (receptor
sites 1-5 (Catterall, 1986), and receptor site 6 (Fainzilber et al., 1994)) and two unidentified (CsTx ()and GPT
(Catterall, 1992)) receptor sites on the rat brain sodium channel and
have been used as tools for functional mapping and characterization of
the channel (Catterall, 1986, 1992; Fainzilber et al., 1994).
Over the past decade a number of selective toxin ligands have been characterized that compete directly on their binding but by various criteria cannot share precisely the same receptor binding sites (Adams and Olivera, 1994; Gordon et al., 1992). Receptor sites are thought to overlap when they appear to be targeted by a number of different toxins that induce similar pharmacological effects and compete in binding assays. Complexes of such overlapping sites have been termed macrosites (Olivera et al., 1991). We suggest that a receptor site be defined as the combined points of attachment/recognition sites that are directly involved in the binding interactions with a toxin. A macrosite, in this context, may include distinct receptor sites for several toxins, each with its own unique points of attachment. Some of these recognition sites may be shared by different toxins that bind in the same macrosite. For example, receptor site 1 on the sodium channel (Catterall, 1986, 1992) binds the blockers tetrodotoxin, saxitoxin, and µ-conotoxins. We suggest that this is in fact a macrosite, since tetrodotoxin and saxitoxin do not share all of their attachment points (Terlau et al., 1991). Furthermore, µ-conotoxins selectively bind to skeletal muscle sodium channels (Moczydlowski et al., 1986) on which at least part of their attachment points are distinct from those of saxitoxin (Stephan et al., 1994).
Receptor sites for peptide neurotoxins that inhibit
sodium current inactivation are of particular interest for the study of
the dynamics of channel gating since neurotoxin binding at these
extracellular regions can affect the inactivation process at
intramembranal segments of the channel (Catterall, 1992). These are the
macrosite that binds sea anemone and -scorpion toxins (site 3 of
Catterall(1986)) and the receptor sites for GPT, CsTx, and
TxVIA
(Gonoi et al., 1986, 1987; Fainzilber et al., 1994).
The latter three receptor sites have been shown to be distinct from
each other on the basis of binding and pharmacological studies
(Catterall and Beress, 1978; Ray et al., 1979; Gonoi et
al., 1986, 1987; Fainzilber et al., 1994). Furthermore,
TxVIA has the unique property of distinguishing between phyletic
variants of sodium channels on the basis of activity but not binding
(Fainzilber et al., 1994). Thus subtle differences in the
TxVIA receptor site on different channels may be the cause of
significant differences in toxin effects; and conversely sequence
variabilities in a family of toxins interacting with a single channel
subtype may cause differing agonist/antagonist activities. Therefore,
the aim of this study was to find a peptide homologous to
TxVIA,
which would act as an agonist at its receptor site on the rat brain
sodium channel. Screening of piscivorous Conus venoms revealed
a new conotoxin that competes with
TxVIA on binding to both rat
brain and molluscan sodium channels and acts as a full agonist in rat
brain. This new toxin, designated NgVIA, will serve as a complementary
pharmacological probe for the study of the role(s) and structureof the
TxVIA receptor site and related receptor sites in modulation of
sodium channel gating.
Figure 1:
Purification of NgVIA. A, 50
mg of lyophilized venom was extracted as described under
``Experimental Procedures,'' and separated on a Sephadex G-50
column (63 1.42 cm), equilibrated, and eluted in 0.1 M ammonium acetate, pH 7.5, at a flow rate of 5 ml/h and a
temperature of 4 °C. B, the marked fraction was further
fractionated by reverse phase HPLC on a Vydac C18 column (25
0.46 cm, 5 µm particle size), eluted at a flow rate of 0.5 ml/min
with a gradient of acetonitrile in 0.1% trifluoroacetic acid as shown
by the dashed line. C, the active fraction (indicated
by the arrow in B) was further purified on a Vydac
phenyl column (25
0.46 cm, 5 µm particle size) using the
same flow rate and solvents. The inset shows complete identity
in the superimposed UV spectra sampled by a diode array detector at
different time points along the NgVIA peak. D, synthetic NgVIA
was prepared as described under ``Experimental Procedures,''
and purified to homogeneity on an Alltech C8 column (25
0.46
cm, 5 µm particle size) at a flow rate of 0.5 ml/min, using a
linear gradient of 0-60% acetonitrile, 2-propanol (1:1) in 0.1%
trifluoroacetic acid. The lower trace (1) is pure
synthetic NgVIA, and the upper trace (2) is an equimolar mix
of native and synthetic NgVIA.
After incubation for the designated time periods, the reaction mixture was diluted with 2 ml of ice-cold wash buffer and filtered through GF/F (Whatman, U. K.) under vacuum. Filters were rapidly washed an additional two times with 2 ml of buffer. Nonspecific toxin binding was determined in the presence of 1 µM unlabeled TxVIA and typically consisted of 30-35% of total binding, one-third of which was to the filters alone. Binding data were analyzed using the iterative computer program LIGAND (Elsevier Biosoft, U. K.).
Our primary aim was to identify a small peptide ligand that
might act as an agonist at the -conotoxin receptor site in rat
brain. Preliminary assays on piscivorous Conus venoms revealed
that the venom of C. nigropunctatus contained a <4-kDa
fraction that displaced
TxVIA from its binding sites on both rat
brain synaptosomes and molluscan central nervous system. Therefore this
venom was chosen for further fractionation. Fractions were assayed in
parallel for toxicity to fish and inhibition of
TxVIA binding in
both rat and mollusc neuronal membrane preparations.
The amino acid
sequence of the toxin was determined by gas-phase automated Edman
sequencing after reduction and pyridylethylation. A single unambiguous
sequence of 31 amino acid residues was obtained in two separate runs (Table 1) and was in good correlation with the amino acid
composition analysis of the toxin (Table 2). Interestingly, the
amino acid sequence of the new toxin included a cysteine framework
identical to that of TxVIA, with identical numbers of residues in
the intercysteine loops (Fig. 2). Two other residues are
identical, and there are a number of similarities in the positioning of
hydrophobic residues, although the overall net charges of the peptides
are contrasting (Fig. 2).
Figure 2:
Sequence comparison of conotoxins that
inhibit sodium channel inactivation. Identical residues in all four
toxins are shown in bold type and are boxed. The
standard one-letter code for amino acid residues (except O = trans-4-hydroxyproline) is used. Spacers () are inserted
to show maximal homology.
, net hydrophobicity calculated
according to Fauchere et al.(1988). References for sequences:
NgVIA, this paper; GmVIA, Shon et al.(1994); TxVIA/B,
Fainzilber et al.(1991).
As we were unable to verify the amino acid sequence data by mass spectrometry, the peptide was synthesized with free C-terminal and folded as described under ``Experimental Procedures.'' As expected from such a hydrophobic sequence the final yield of active (i.e. correctly folded) peptide was low, averaging 3 nmol of active toxin/10 mg of crude synthetic peptide (folding efficiencies ranged from 0.1 to 0.5%). The final purified product co-eluted with native NgVIA in reverse phase HPLC (Fig. 1D) and had the same activity as native toxin in electrophysiological tests and in vivo assays in rat brain (see below).
The paralytic activity of NgVIA was examined in
bioassays on fish (Gambusia), snails (Patella), and
fly larvae (Sarcophaga). Although the toxin has potent
paralytic activity on fish (ED = 2.8 pmol/100 mg of
body weight) and snails (ED
= 14.5 pmol/100 mg),
there were no observable effects on fly larvae at doses of up to 250
pmol/100 mg.
Figure 3:
Inhibition of voltage-dependent sodium
current inactivation by TxVIA and NgVIA. Sodium currents at 10 mV
were recorded from caudodorsal neurons of the snail L. stagnalis in the whole cell voltage clamp mode. The effects of 1.2
µM
TxVIA (A), 1 µM venom-derived NgVIA (B), and 0.8 µM synthetic NgVIA (C) are shown. Left panels are
control currents, middle panels show currents recorded in the
presence of the toxin, and right panels show currents recorded
after 1 min of wash out of the toxin. The data shown are representative
results from a number of different cells. Capacitive transients were
clipped in the illustrations. Calibration bars are 50 ms and 0.5
nA.
Figure 4:
Pharmacology of NgVIA in rat brain and
snail central nervous system membrane preparations. A,
inhibition of I-
TxVIA binding by NgVIA. Neuronal
membranes were incubated with 0.2 nM
I-
TxVIA and increasing concentrations of NgVIA.
The amount of
I-
TxVIA bound at each data point is
expressed as a percentage of the maximal specific binding in the
system. Circles,Helix central nervous system
(IC
= 59.4 ± 11.4 nM), full
triangles, rat brain synaptosomes (IC
= 4.9
± 1.2 nM, empty triangles, lysed rat brain
synaptosomes (IC
= 18.7 ± 4.4 nM). B, NgVIA and
TxVIA effects on
Na influx in
rat brain synaptosomes. Synaptosomes were incubated with toxins as
described under ``Experimental Procedures,'' and net influx
of
Na after 30 s was determined. Enhancement of
veratridine (Ver)-induced flux examined at 2 µM veratridine and 1 µM
TxVIA or NgVIA. Results are
shown as a percentage of the control flux induced by 2 µM veratridine (1.2
0.3 nmol of sodium/min/mg of
protein). The maximal flux obtainable is shown by the rightmost bar (effect of 200 µM veratridine).
The effects of
NgVIA on rat brain sodium channels in vitro were examined by Na influx assays in rat brain synaptosomes. NgVIA alone
was not able to initiate sodium influx (Fig. 4B), in
common with other inactivation-inhibiting toxins (Catterall and Beress,
1978; Fainzilber et al., 1994). However, NgVIA synergically
increased the veratridine-stimulated uptake of
Na to
approximately 3-fold above control levels (Fig. 4B).
This is similar to the effect previously obtained with CsTx in this
system (Fainzilber et al., 1994).
Figure 5: Effects of NgVIA in rat brain in vivo. A, rats were injected intracranially with NgVIA, and their reactions were followed for up to 20 min postinjection. Circles, 100 pmol of NgVIA (n = 2); triangles, 40 pmol of NgVIA (n = 3); squares, 20 pmol of NgVIA (n = 3). B, upon simultaneous injection of 40 pmol of NgVIA with 20 nmol of TxVIA (full triangles, n = 3), the full repertoire of toxic symptoms was seen without very marked change relative to the control of 40 pmol of NgVIA alone (empty triangles). However, when 20 nmol of TxVIA were injected 20 min before administration of 40 pmol of NgVIA (full diamonds, n = 4), the toxic effects were clearly delayed and reduced.
Application of lethal doses of synthetic NgVIA to rats revealed a similar progression of symptoms to that seen with the venom-derived toxin. The rats underwent rapid paralysis and commenced seizures, with death occurring within 10 min. A sublethal dose caused transient hyperactivity and pronounced shaking movements, as seen previously with native NgVIA.
In the present study we have identified and characterized a novel peptide conotoxin that affects sodium channel inactivation. As will be detailed below this toxin provides an important complement of the pharmacological tools required for understanding the functional role of different receptor sites in gating mechanisms of sodium channels.
There are also qualitative differences in the
effects of the two toxins on the sodium current, for example the
increase of peak sodium current by NgVIA (Fig. 3). The partial
antagonistic effects of TxVIA versus NgVIA might be
explained by postulating a higher efficacy of NgVIA activity and/or
differences in their binding kinetics. Another possibility is that
NgVIA identifies an additional minor population of sodium channels in
rat brain, which is not recognized by
TxVIA. The lower capacity of
TxVIA receptors in rat brain synaptosomes (Fainzilber et
al., 1994) as compared with saxitoxin receptors (Ray et
al., 1978) is consistent with this possibility. These aspects
should be examined in the future when labeled analogs of NgVIA become
available.
Binding of NgVIA appears to be at least partially
voltage-dependent (Fig. 4A) as is that of CsTx (Gonoi et al., 1987) and the other toxins that inhibit sodium current
inactivation. Depolarization of the membrane by lysis of synaptosomes
causes a 4-fold increase in the IC for inhibition of
I-
TxVIA binding by NgVIA. This is comparable with
the 5-fold increase in K
for sea anemone toxin
II action in depolarized neuroblastoma cells (Catterall and Beress,
1978).
The similarity in binding characteristics and interactions of
NgVIA with TxVIA in vitro and in vivo to those
reported for CsTx (Fainzilber et al., 1994), both
qualitatively and quantitatively, suggests that they may bind to
closely related sites (see below and Fig. 6). Thus the IC
values of both toxins on both rat and mollusc neuronal
preparations are similar, as is their synergic effect with veratridine
on sodium flux. It is tempting to suggest that NgVIA may have some
attachment points in common with those of CsTx; however, the
differences between them as regards antagonism in vivo by
TxVIA ( Fig. 5and Fainzilber et al.(1994)) suggest
that their receptor sites are probably not identical. These differences
might also be attributable to different kinetics of binding or efficacy
in action of these two toxins. It will be of interest to study these
possibilities in the future by direct binding experiments with NgVIA.
Figure 6:
A
model visualizing locations of peptide neurotoxins affecting sodium
current inactivation, bound to their putative receptor sites. The
sodium channel extracellular surface is shown from above, with the four
homologous repeat domains represented by shaded outlines (I-IV). A, summary of the binding inhibition among the
different peptide neurotoxins. The arrows are approximately
proportional to the inhibition caused by each toxin on the binding of
radiolabeled -scorpion toxin (
ScTx) or
TxVIA in
rat brain synaptosomes. B, illustration of the peptide toxins
bound at their putative receptor sites. The receptor site for the
sodium channel blocker tetrodotoxin (TTX) has been localized
to the extracellular vestibule of the ion-conducting pore (Terlau et al., 1991) and is indicated in the center of the
model to facilitate orientation. See text for explanations. ATXII, sea anemone toxin II.
NgVIA is unique in the group of toxins
shown in Fig. 2in that it is the only one so far found with
significant activity in vertebrates. Indeed it is more potent than CsTx
in rat brain by 1 order of magnitude (compare Fig. 5with
Fainzilber et al.(1994)). The potent paralytic activity of
NgVIA on rats and molluscs and its effects on sodium currents in rat
brain synaptosomes and Lymnaea neurons indicate that NgVIA is
an agonist of both mammalian and molluscan sodium channels. However,
NgVIA cannot be considered a wide range cross-phyletic agonist since it
has no discernible paralytic activity on insects. It seems that changes
in the composition of the intercysteine loops on a conserved
-conotoxin framework may cause significant differences in activity
on one variant of sodium channels (i.e. in rat brain), while
relatively little change is observed for another variant (i.e. in mollusc central nervous system). NgVIA should be a valuable
reference for structure-function analyses of this group of toxins,
since it enables a rational analysis of the structural elements
necessary for agonist activity of these toxins on the rat brain sodium
channel. However, the primary structure variability in this group (Fig. 2) suggests that three-dimensional structural data will be
necessary to tackle this question.
For clarity of presentation, we have placed the
toxins that do not bind to macrosite 3 (CsTx, GPT, TxVIA) in
different domains of the channel. GPT competes with both
-scorpion
toxin (Gonoi et al., 1986) and
TxVIA (Fainzilber et
al., 1994) but at concentrations 25-fold higher than its K
on mammalian neurons, suggesting that it binds
to a distinct site. Therefore GPT is positioned in proximity to both to
allow steric interference that may cause the competition between them.
CsTx, in contrast, competes at very low concentrations with
TxVIA
and is completely antagonized by
TxVIA in rat brain (Fainzilber et al., 1994). Therefore the receptor sites of CsTx and
TxVIA are proposed to partially overlap (Fig. 6B)
but are not identical since these two toxins reveal opposite allosteric
interactions with alkaloid toxins bound at site 2, such as
batrachotoxin and veratridine (Gonoi et al., 1987; Fainzilber et al., 1994).
The partial protection observed by
administration of TxVIA prior to NgVIA in rat brain in vivo (Fig. 5) is in contrast to the complete protection or
antagonism observed by simultaneous injection of CsTx and
TxVIA
(Fainzilber et al., 1994). This suggests that the NgVIA and
TxVIA receptor sites are partially overlapping, similar to CsTx
and
TxVIA, but may share different points of attachment (Fig. 6B). It should be noted, however, that steric
interference (with no overlap) cannot be excluded (Gordon et
al., 1992; Moskowitz et al., 1994). The interaction of
NgVIA with the
TxVIA receptor site is very similar to that of CsTx
(see above). Moreover, the toxic effects of NgVIA are at least
partially antagonized by the presence of
TxVIA (Fig. 5). On
this basis we suggest that the receptor site of NgVIA is partially
overlapping with both the CsTx and
TxVIA receptor sites (Fig. 6B). The similarity in the binding properties
(dependence on membrane polarization, competition with
TxVIA, and
interaction with veratridine) and activity in vivo between
NgVIA and CsTx suggest substantial overlap in their receptor sites.
The model presented in Fig. 6gives a graphic visualization
of the different peptide toxins bound to their putative receptor sites
on the outer surface of sodium channels while emphasizing the lack of
structural information on the molecular level on these receptor sites.
All -conotoxins inhibit sodium channel inactivation and as
exemplified by NgVIA and
TxVIA may do so via binding at distinct
receptor sites. Localization of the attachment points comprising these
receptor sites may shed light on the mechanism of action of toxins that
modify sodium channel gating. The use of
TxVIA and NgVIA as
pharmacological sensors for minor differences in sodium channel
variants provides a rational approach to this complex problem and may
contribute to the elucidation of structure-function relationships in
sodium channels.