(Received for publication, May 18, 1995; and in revised form, August 3, 1995)
From the e 10, 2300 Kiel, Federal Republic of
Germany, and the
New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of I-dendrotoxin I (a classical ligand for K
channel) to rat brain membranes and behave as blockers of
voltage-sensitive K
channels. Sea anemone kalicludines
are 58-59-amino acid peptides cross-linked with three disulfide
bridges. They are structurally homologous both to dendrotoxins which
are snake venom toxins and to the basic pancreatic trypsin inhibitor
(Kunitz inhibitor) and have the unique property of expressing both the
function of dendrotoxins in blocking voltage-sensitive K
channels and the function of the Kunitz inhibitor in inhibiting
trypsin. Kaliseptine is another structural class of peptide comprising
36 amino acids with no sequence homology with kalicludines or with
dendrotoxins. In spite of this structural difference, it binds to the
same receptor site as dendrotoxin and kalicludines and is as efficient
as a K
channel inhibitor as the most potent
kalicludine.
Potassium channels have an essential role in repolarization
phases of action potentials and in the fine regulation of the resting
potential. Molecular cloning has recently led to the identification of
a large number (over 15) of genes for voltage-sensitive, non
inward-rectifier, K (Kv) channels (1, 2) which, when expressed in Xenopus oocytes, generate a variety of K
channel
activities with different kinetics, voltage dependences, conductances,
and regulation properties. Surprisingly, only a relatively small number
of toxins active on these channels has yet been
discovered(3, 4) . They are MCD peptide from bee
venom(5, 6) , charybdotoxin and analogs from different
scorpion species (7, 8, 9, 10, 11, 12, 13, 14) ,
-bungarotoxin(15, 16) , and dendrotoxins from
mamba venoms (3, 5, 17, 18, 19, 20, 21, 22) .
These different toxins only block the expression of four of the cloned Kv channels (Kv1.1, Kv1.2, and Kv1.6 for MCD peptide and dendrotoxin, Kv1.1, Kv1.2, Kv1.3, and Kv1.6 for charybdotoxin) (reviewed in (23) ). Binding studies using radioiodinated derivatives of these toxins have been essential for the identification, purification, and determination of the subunit structure (6, 24, 25, 26) of these Kv channels. These toxins have also been important for the first brain localizations of Kv channels (16, 27) and are particularly interesting inducers of long term potentiation(28) .
Sea anemones produce toxins with which
they paralyze their prey. They are particularly important as sources of
toxins active on voltage-dependent Na channel which
have been essential tools for studying the structure, the mechanism,
and the diversity of this channel
type(29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .
This paper reports the isolation, structure, and properties of a series of new toxins from Anemonia sulcata which behave as blockers of Kv channels.
Figure 2:
Sequence homologies of A. sulcata kalicludines with DTX (top) and the basic
pancreatic trypsin inhibitor, BPTI (bottom).
Figure 1:
A, purification
on SP Sephadex C-25 of 6 g of crude sea anemone toxic material obtained
from A. sulcata as described under
``Experimental Procedures.'' The column (2.5 140 cm)
was equilibrated with 0.01 M ammonium acetate buffer, pH 4.5,
the crude toxic fraction was loaded and eluted in this buffer, followed
by a stepwise gradient as indicated in the figure. The final elution
was carried out with 1 M sodium chloride (not shown). Elution
was monitored by measuring the absorbance at 278, flow rate, 225 ml/h.
Fraction size, 8 ml. Collected fractions 30, 34, and 38 are shaded; B-D, last purification step of AsKS,
AsKC1, and AsKC2, respectively, as described under ``Experimental
Procedures.'' Collected fractions are shaded. E,
last purification step of AsKC3. The peptide is indicated by the arrow. F, elution profile of AsKC3 in the same
conditions as in E.
Solvents used for HPLC were a linear gradient
between solution A = 1% acetic acid and solution B = 1 M ammonium acetate passed in 50 min at a flow rate of 1 ml/min
for the cation exchanger column TSKSP 5PW (7.5 75 mm), and
different gradients between solution C = 0.5% trilfluoroacetic
acid plus 0.85% triethylamine, plus 10 µl/liter
-mercaptoethanol in water, and solution D = the same
components in acetonitrile, for the three different RP18 reverse phase
columns used. At each chromatographic step, all the eluted fractions
were checked for their ability to inhibit
I-DTX
binding to its receptor in rat brain P3 membranes. After the last
purification step, pure peptides were lyophilized and desalted on RP18
Lichrocart) with mixtures of 0.1% trifluoroacetic acid in water and
acetonitrile. The peptides were first absorbed in 2% acetonitrile and
then eluted with 50% acetonitrile and lyophilized.
Figure 3: Sequence homologies of AsKS with the sea anemone B. granulifera (BgK) toxin.
We have purified from the sea anemone A. sulcata four peptides for their ability to prevent I-DTX
binding to its receptors in rat brain.
Since DTX
is a well-known blocker of K
channels and since these A. sulcata peptides not only
inhibit
I-DTX
binding but also inhibit
K
channel activity it has been decided to designate
them under the name of A. sulcata kaliseptine and kalicludines
(AsKS and AsKC). Fig. 2and 3 present the sequences of the four
peptides. Three of them have sequence similarities with DTX
and the basic bovine trypsin inhibitor (Kunitz inhibitor, BPTI)
which is a well-known homolog of DTX
(17) . These
toxins were named A. sulcata kalicludines (AsKC1, AsKC2, and
AsKC3) because they also have structural homologies with calcicludine
(CaC) (32-35%), another homolog of the Kunitz inhibitor and of
DTX
(43, 46) which is a blocker of
voltage-sensitive Ca
channels. The last purified
peptide (Fig. 3) is not homologous to DTX
. It was
named A. sulcata kaliseptine (AsKS).
Fig. 4shows
the concentration dependence of the inhibition of I-DTX
binding by the different sea anemone
toxins. Values of IC
are 27 nM for AsKS, 60
nM for AsKC2, 375 nM for AsKC1, and 500 nM for AsKC3. These values were 2-4 orders of magnitude higher
than the IC
for DTX
inhibition of
I-DTX
binding which is 0.14 nM.
Scatchard plots presented in Fig. 5show that both kaliseptine
(AsKS) and kalicludines (AsKC2) inhibit
I-DTX
binding in a competitive way. Plots of the apparent values K
(app)/K
versus the
toxins concentrations provide the true values of inhibition constants K
which are 10 nM for AsKS and 20 nM for AsKC2 (Fig. 5, insets). None of the three
kalicludines (nor AsKS) prevented
I-calcicludine (
)binding to its receptors in rat brain up to the
concentration of 5 µM, although they are also structurally
homologous to this Ca
channel blocking toxin (46) .
Figure 4:
Inhibition by DTX, the Kunitz
trypsin inhibitor, and different peptides from A. sulcata of the specific
I-DTX
binding to rat
brain microsomes. Unlabeled DTX
and the different peptides
were first incubated at different concentrations with the membranes (20
µg/ml) and then
I-DTX
(3 pM) was
added and membranes were incubated for 1 h at 25 °C. Results are
mean of two experiments. Nonspecific
I-DTX
binding was below 2% and was subtracted.
, AsKS;
,
AsKC1;
, AsKC2;
, AsKC3;
, DTXI;
, the Kunitz
inhibitor BPTI.
Figure 5:
Binding of I-DTX
to rat brain membranes in the presence of different
concentrations of AsKS (A) and AsKC2 (B). Membranes
(7 µg of protein/ml) were incubated for 1 h at 25 °C with
I-DTX
(2-100 pM). Main
panels, Scatchard plots for
I-DTX
-specific binding obtained after
subtraction of nonspecific
I-DTX
binding
determined by including 0.1 µM DTX
in the
incubation medium. Results are means of two determinations. Bound/free
is expressed as pmol/(mg of protein
nM). A,
concentrations of AsKS:
, 0 nM;
, 2 nM;
, 8 nM;
, 27 nM. B,
concentrations of AsKC2:
, 10 nM;
, 30
nM;
, 60 nM. Insets, effects of
increasing concentrations of A. sulcata peptides on
the K
(app)/K
ratio where K
(app) is the
value obtained in the presence of toxins.
Electrophysiological measurements presented in Fig. 6show that these peptides from A. sulcata which recognize DTX receptors also inhibit the Kv1.2
K
channel expressed in Xenopus oocytes as
DTX
does. The IC
for inhibition of the Kv1.2
current ranged from 140 nM for AsKS to 1.1 µM for
AsKC2 to 1.3 µM for AsKC3 to 2.8 µM for
AsKC1. Under these experimental conditions, DTX
itself has
an IC
of 2.1 nM. Peptides which inhibit the Kv1.2
current with the best efficiency are also those which have the highest
affinity for the DTX
receptors.
Figure 6:
Inhibition of the K
current in Xenopus oocytes expressing the Kv1.2 channels. The
oocytes were injected with 0.2 ng of Kv1.2 cRNA. In these experiments,
the holding potential was -80 mV, and current amplitudes were
measured at +30 mV (n = 3). The inset shows current traces recorded in control and in the presence of
AsKS peptide (600 nM).
, AsKS;
, AsKC1;
,
AsKC2;
, AsKC3;
, DTX
;
,
BPTI.
Since the three
kalicludines have extensive homologies with BPTI which is a very potent
blocker of trypsin activity, it was checked whether kalicludines also
could have an ability to inhibit trypsin. Trypsin at the concentration
of about 3 µM was first incubated at room temperature for
3 h with different concentrations of the kalicludines to be sure to
reach equilibrium. After incubation, the free trypsin was measured by
its ability to release paranitroaniline from BAPNA. Fig. 7shows
that all three kalicludines inhibit trypsin. Inhibition profiles of
trypsin by AsKC1, AsKC2, AsKC3, and BPTI are very similar. Total
inhibition of trypsin was reached by addition of a stoichiometric
amount (1:1) of the kalicludines. The profile of inhibition observed
with AsKC1, AsKC2, and AsKC3 (which indicates a stoichiometric 1:1
inhibition) is encountered when the K for the
interaction is below 1/100th of the concentration of trypsin in the
incubation medium(47) , here 3 µM. Then, the K
of interaction of these sea anemone peptides
with trypsin has to be below 30 nM. Conversely, Fig. 7also shows that a large excess of DTX
, another
Kv channel inhibitior of similar structure, is unable to inhibit
trypsin. This lack of trypsin inhibition was also observed for toxin K
from D. polylepis, another dendrotoxin toxin for
K
channels (17) which is structurally more
closely related to the Kunitz inhibitor than DTX
because it
has a lysine in position 15 corresponding to the essential lysine 15 at
the active site of the trypsin inhibitor (48) (the
corresponding residue is a tyrosine in DTX
). Finally, Fig. 7shows that A. sulcata kaliseptine AsKS
which has no structural homology with BPTI does not inhibit trypsin
even at a molecular excess of 7 to 1.
Figure 7:
Concentration dependence of trypsin
inhibition by different concentrations of BPTI, A. sulcata peptides, and dendrotoxins. , AsKS;
, AsKC1;
,
AsKC2;
, AsKC3;
, DTX
;
, toxin K from D. polylepis.;
, BPTI. Trypsin activity is
plotted against the molecular ratios of the different peptides to
trypsin in the incubation medium.
The first category of peptides isolated in this work has been
designated A. sulcata kalicludines. They inhibit I-DTX binding to Kv channel proteins and block the Kv1.2
channel expressed in the Xenopus oocyte. Sea anemone
kalicludines 1, 2, and 3 are 57-60 amino acid peptides homologous
to serine protease inhibitors of the Kunitz type and to dendrotoxins (Fig. 2). They have from 40 to 41% homologies with the Kunitz
inhibitor and from 38 to 42% homologies with DTX
. These
sequence homologies led us to compare more closely their properties
with those of these two peptides. DTX
is the most potent
blocker of the Kv1.2 channel(42) . Affinity for its receptor in
rat brain is very high,
10
M as also
shown in Fig. 4. On the other hand, BPTI is the most potent
inhibitor of trypsin with a dissociation constant of
6.10
M(47) but is not a blocker
of Kv channels.
Fig. 4, Fig. 6, and Fig. 7show
that A. sulcata kalicludines are molecules with dual
types of activity. They have both the properties of DTX and
BPTI, they prevent
I-DTX
binding to Kv1.2
channel in a competitive manner (Fig. 5B), and they
inhibit trypsin in a stoichiometric manner like BPTI. From titration
curves presented in Fig. 7, one can deduce that K
of these trypsin inhibitions is below 30 nM. Conversely
and as expected, the other class of sea anemone K
channel toxins, kaliseptine, which is structurally different from
the Kunitz-type proteinase inhibitors, does not inhibit trypsin. It is
the first time that both functions, i.e. blockade of a
K
channel and a potent inhibition of a protease, have
been found in a single molecule of about 60 amino acids and
structurally related to BPTI. Dendrotoxins do not inhibit trypsin, and
protease inhibitors do not display dendrotoxin-like activity even at
high concentrations(49) .
Sea anemones appeared at least 800
millions years ago and it may be that bifunctional molecules such as
kalicludines are survivors of a remote past, and that evolution has
gradually given rise, for efficiency, to molecules fully specialized
either for trypsin inhibition or for blockade of the voltage-sensitive
K channel.
The molecular structure basement of
dendrotoxins and BPTI is shown in Fig. 8a. This
basement corresponds to pear-shaped molecules of 35 Å length
and 25 Å of grand diameter. The replacement of the peptide
sequence of AsKC2 in the coordinates of the mean NMR structure of
DTX
only induced a few unfavorable non-bonded atomic
contacts between side chain atoms of Asn
and
Asp
, Arg
and Arg
, Lys
and Asn
. All could be removed by the only change of
the
angle by rotation of the side chain around
C
-C
bonds of Asn
,
Arg
, and Lys
. All the residues forming the
hydrophobic interior of both BPTI and DTX
are well
conserved and could be easily adapted at the same place. These amino
acids are tyrosine at positions 21, 22, 23, and 35, phenylalanine at
positions 33 and 45. AsKC2 (net charge of +8) is less charged than
DTX
(+11) and more charged than BPTI (+6) (Fig. 8b).
Figure 8:
Main chain folding of the
averaged-minimized NMR structure of dendrotoxin I drawn using MOLSCRIPT (53) and the 1DEM entry of the Protein Data
Bank(43, 45) . Disulfide bridges are represented in a
``ball-and-stick'' style. The trypsin binding site in BPTI is
located in the loop comprizing residues 15-19 in dendrotoxin I. = NH
-terminal position, Ct = COOH-terminal position 60. b, space-filling
atomic models of (left to right in each panels) AsKC2, dendrotoxin I
and BPTI. Basic amino acids Arg, His, and Lys are colored in blue, acidic amino acids Asp and Glu in white, and
the others in red. In the upper left panel, the
models are shown in the same orientation as in a. In the upper right panel, the structure were rotated by 90 °C and
are shown on the Ct and Nt side. The lower left panel is
obtained by a further 90° rotation and shows the opposite side of
the orientation given in a (up-and-down). The lower right
panel is an additional rotation by 90° showing the apical
trypsin binding side described in a.
A recent analysis explaining why
dendrotoxins are unable to inhibit trypsin has ben given(50) .
Replacement of both the Lys of BPTI by a Tyr, and
Ala
by a longer amino acid (aspartate in dendrotoxin
and glutamine in DTX
), produce a highly unfavorable complex
with the trypsin active site. Moreover, the presence in DTX
of a proline in position 21 that is occupied by an isoleucine in
BPTI has been seen as an additional unfavorable feature for good atomic
contact with Tyr
of trypsin. Among these three positions
necessary for a good inhibition of trypsin by BPTI, two central ones,
Arg
and Ala
, are conserved in AsKC2.
Pro
which is the equivalent to Pro
in
dendrotoxins is retained. Thus, the prediction would be that AsKC2 has
indeed, as it is found, a trypsin inhibitory property but with an
efficacy which would be lower than that of BPTI.
It has been
recently proposed (43) that the most prominent structural
difference between BPTI and dendrotoxins concerned surface
electrostatics which could explain differences between the two types of
molecules in their capacity to block K channels. The
space-filling models presented in Fig. 8b show that the
major surface-electrostatic differences existing between DTX
and BPTI in the COOH-terminal and NH
-terminal regions
(that are packed together due to the peptide fold) are in part
reproduced when comparing AsKC2 and BPTI. For instance, the cluster
Arg
, Arg
, Arg
, and Lys
in DTX
is replaced by another four-charges cluster
made of Lys
, Lys
, Lys
, and
Arg
in AsKC2 (upper right panel of Fig. 8b). Negative charges in the COOH-terminal
-helix are slightly modified in AsKC2 relative to DTX
with a Glu
-Cys
-Glu
sequence
instead of Glu
-Glu
-Cys
. The
negative charge on Glu
in DTX
I is conserved
in AsKC2 (Glu
) as well as the positive charge on Lys
of DTX
(Lys
in AsKC2).
The structure
of kaliseptine (AsKS) is different from that of kalicludines. It is
also a much smaller peptide (only 36 amino acids). It has no homology
with proteinase inhibitors of the Kunitz type but presents 49%
homologies with a 37-amino acid peptide isolated from another sea
anemone, Bunodosoma granulifera (BgK, Fig. 3)
which is also a probable blocker of the voltage-dependent K channel(s) sensitive to DTX
since it inhibits
I-DTX
binding(51) . However these two
structures have a striking difference in the position of one of their
Cys residues (Cys
and Cys
in the
COOH-terminal part). This difference is surprising since Cys residues,
as for all other short polypeptide toxins, are expected to form
disulfide bridges. It might result from a mistake in the sequence of
the B. granulifera peptide. Both AsKS and BgK also
have limited homologies with a 110-amino acid protease inhibitor from
the eggwhite of a red sea turtle (51, 52) which
itself has some homologies with BPTI. However homologies with AsKS are
limited to the sequence 58-93 of the sea turtle inhibitor and not
to the sequence 3-58 which is homologous to BPTI. There could
exist other homologs of kaliseptines with a trypsin inhibitor activity
but they have not been identified yet.
DTX blocks with a
high affinity not only Kv1.2 channels but also Kv1.1 and Kv1.6 channels
when they are expressed in Xenopus oocytes as well as channels
formed by heterologous association of different subunits corresponding
to these different types of channels(1, 2) . Therefore
measuring the capacity of sea anemone kalicludines and kaliseptines to
inhibit
I-DTX
binding to neuronal membranes
is not equivalent to measuring their capacity to inhibit oocyte
expression of a particular class of K
channel (in this
paper Kv1.2).
I-DTX
binding identifies a
multiplicity of types of voltage-sensitive K
channels(14, 23, 24) . For all these
reasons a very systematic study of the different toxins described in
this work will have to be made on different types of Kv channels, as
previously done for DTX
(23) . This will require
larger quantities of these peptides which are in relatively low amount
but could now be prepared by total synthesis or in a recombinant way.
Also, it has been shown before that a particularly interesting
property of sea anemone venom is that it not only contains toxins that
can disciminate between different types of voltage-sensitive
Na channels in different tissues of a given animal but
also between Na
channels in different animal
species(35, 37) . A good number of the sea anemone
toxins for Na
channels have only a very small activity
toward mammals but a very high activity toward
crustaceans(40) . When larger quantities of the peptides
described in this work become available, it will be particularly
interesting to assay their activity in crustaceans and also in insects
which often have nervous system properties similar to those of
crustaceans. From a biological point of view it would make sense that
sea anemones make toxins more oriented toward K
channels normally found in their preys (fish, crustaceans, etc.).
On the other hand, if these new peptides, or analogs of these new
peptides, were particularly active on insects, this could provide new
ways of thinking toward the development of new insecticides.
Finally
the mixture of ion channel toxins found in sea anemone venom and
comprising both Na channel toxins tending to
``activate'' the Na
channels (37) and K
channel toxins tending to block
K
channels is expected to have devastating neurotoxic
effects by producing very massive release of neurotransmitters as well
as very potent effects on heart, muscle, and endocrine cells.