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Research Communications |
Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milano, Italy; Department of General Physiology and Biochemistry, Laboratory of Electrophysiology, University of Milano, 20133 Milano, Italy;
* Department of Molecular Recognition and Structural Biology, Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Morelos 62271, Mexico; and
Institute of General Pathology, University of Florence, I-50134 Firenze, Italy
2Correspondence: Dipartimento di Fisiologia e Biochimica Generali, Via Celoria 26, 20133 Milano, Italy. E-mail: enzo.wanke{at}unimi.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: eag gene family • erg gene • ergtoxin • scorpion toxin • action potential
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6
7)
for the first
purifications, structural analyses, localization, and identification of
pore-forming regions of voltage-dependent K+
channels, which are the most numerous and diversified of ion channels
(8)
.
The ether-a-go-go-related genes
(erg)3
(9,
10)
are well expressed in heart (11)
, peripheral sympathetic
ganglia (10)
, brain (12)
, and tumor cells (13
14
15)
, and codify for a
K+ channel (ERG) that is unusual insofar as its
current (IERG) shows a form of inward
rectification apparently similar to that of the inward rectifier
channels (two transmembrane segments), but has the six putative
transmembrane segments typical of depolarization-activated channels.
Since the discovery (16,
17)
that its heterologous expression encodes a
K+ current that plays a fundamental role in heart
physiology (IKr) (18,
19)
and that
mutations of this gene are responsible for the chromosome 7-linked long
QT syndrome (LQT2) (11)
, it has been shown that it may play a crucial
role in various mammalian tissues (20
21
22)
and Drosophila
`seizure' mutant (23,
24)
. Although such different drugs
as class III antiarrhythmics, antihistaminics, and antipsychotics
(haloperidol) are mainly irreversible, use-dependent ERG channel
blockers (25
26
27
28)
, there is need for a specific tool to develop studies
aimed at the definition of the physiological role of ERG.
We describe the isolation and primary structure of a peptide called
ergtoxin (ErgTx, because it is a toxin specific for ERG channels),
which is entirely different from the other 10 subfamilies of known
scorpion toxins (7,
29,
30)
. This toxin is specific for ERG channels in
nerve, heart, and endocrine cells, and is unable to block other
K+ channels, including the most closely related
EAG channel (9,
31)
. This is the first description of a selective toxin
for one member of the eag superfamily; we propose the same
procedures for finding toxins selective for other members of the family
that lack effective blockers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Fraction 8 from CM-cellulose column contained peptides active on ERG
channels and was subsequently separated by two steps of
high-performance liquid chromatography (HPLC). The first used a C18
semipreparative column, with a linear gradient of solution A (0.10%
trifluoacetic acid in water) to solution B (acetonitrile in 0.12%
trifluoroacetic acid) run up to 50% B, in 60 min. Component eluting at
33.86 min was finally separated, using a C18 analytical column run with
a linear gradient from 5% to 40% B in 90 min. Component eluting at
47.2 min was pure ergtoxin.
Sequence determination
A Beckman LF3000 Protein Sequencer was used for this study.
Direct sequencing permitted the identification of the first 30 amino
acid residues. The carboxyl-terminal segment and the overlapping
sequences were unequivocally positioned after sequencing fragments of
ergtoxin. Reduced and carboxymethylated toxin was submitted to direct
sequencing in order to confirm the cysteinyl residues, and was used for
enzymatic digestion with Staphylococcus aureus protease V8.
Another batch of 50 µg was cleaved with cyanogen bromide (32)
. The
peptides were separated by HPLC by using the same C18 analytical column
described above and then sequenced. A protease V8 peptide gave the
overlapping sequence from positions Cys20 to
Cys39, whereas the last segment produced by
cyanogen bromide confirmed the sequence from residue
Met35 to Ala41. The last
amino acid, Pro42, was obtained on the basis of
amino acid composition and mass spectrometry data.
Cell culture
Cells of the F-11 clone (mouse neuroblastoma N18TG-2 x rat DRG)
(33)
and the human neuroblastoma SHSY5Y (Prof. G. Tarone, Department of
Genetics, Biology and Medical Chemistry, University of Turin) were
routinely cultured in Dulbecco's modified Eagle medium (DMEM),
containing 4.5 g/l of glucose and 10% of fetal calf serum (FCS). The
cells were incubated at 37°C in a humidified atmosphere with 5%
CO2. The MMQ pituitary cell line (kindly provided
by Dr. I. S. Login, University of Virginia School of Medicine,
Charlottesville, Va.) was cultured in RPMI 1640 medium containing 7.5%
horse serum and 2.5% FCS. The RINm5F pancreatic insulinoma cell line
was cultured in RPMI 1640 medium containing 10% FCS. The C2C12 clone
was cultured in DMEM medium containing 10% FCS. The human pancreatic
ß cells (kindly provided by Dr. P. Marchetti, Department Metabolic
Diseases, Pisa, Italy) were cultured in Medium 199 (Hyclone Lab. Logan,
Utah) containing 10% ABS.
Cardiac myocytes
Guinea pig ventricular myocytes were freshly dissociated
according to a previously described procedure (34)
. Delayed rectifier
K+ currents were activated by step
depolarizations from a holding potential of -40 mV. The
dihydropyridine nisoldipine (0.2 µM) was added to all of the
solutions to minimize contamination by Ca2+
currents. The rapid (IKr) and slow
(IKs) components of the delayed rectifier current
were dissected by means of voltage protocols and use of the channel
blockers, E-4031 (specific for IKr) and chromanol
293B (specific for IKs) (35)
. Indeed, currents
sensitive to E-4031 and chromanol can be safely assumed to reflect
IKr and IKs respectively.
In evaluating IKs, IKr
contamination was further removed by adding 5 µM E-4031 to all
solutions.
Solutions
For neuronal cells, the standard extracellular solution
contained (mM): NaCl 130, KCl 5, CaCl2 2,
MgCl2 2, HEPES-NaOH 10, D-glucose 5, and pH 7.40.
In the high K+ external solution
([K+]o=40 mM), NaCl was
replaced by an equimolar amount of KCl. The standard pipette solution
at [Ca2+]i =
10-8 M (pCa 8) contained (mM):
K+-aspartate 130, NaCl 10,
MgCl2 2, CaCl2 1.3,
EGTA-KOH 10, HEPES-KOH 10, ATP (Mg2+ salt) 1,
pH = 7.30. For the cardiac cells, the standard extracellular
solution contained (mM): NaCl 154, KCl 4, CaCl2
2, MgCl2 1, HEPES-NaOH 5, D-glucose 5.5, and pH
7.35. The pipette solution contained (mM)
K+-aspartate 110, KCl 23,
CaCl2 0.4, EGTA-KOH 1 (pCa = 7),
MgCl2 3, HEPES-KOH 5, GTP
(Na+ salt) 0.4, ATP (Na+
salt) 5, creatine phosphate 5, pH = 7.3. ErgTx was added to the
extracellular solutions from a stock solution in distilled water.
During the experiments to isolate
IEAG, we omitted 1.3 mM
CaCl2 from the internal solution in order to
buffer it to [Ca2+]i =
10-9 M (pCa 9) and prevent the
Ca2+-dependent block of
IEAG. In some experiments, the data
were corrected by using WAY-123,398 (25)
(WAY). WAY, which was received
from Dr. W. Spinelli (Wyeth-Ayerst Research, Princeton, N.J.), was
dissolved in distilled water to make 10 mM stock solutions. Chromanol
293B was dissolved in DMSO, nisoldipine in ethanol, and E-4031 in water
stock solutions. Ethanol and DMSO at a final concentration <
0.1% were included in the control and the test solutions. Nisoldipine,
E-4031, and chromanol 293B were generous gifts from Bayer
Pharmaceuticals (Milano, Italy), Sanofi Recherche (Montpellier,
France), and Hoechst Marion Roussel (Frankfurt, Germany), respectively.
Patch-clamp recordings
The neuronal currents were recorded at room temperature as
described previously (13)
, but the measurements of cardiac cells
required temperatures in the physiological range (36±0.5°C). Pipette
resistance (4–6 M
), cell capacitance and series resistance errors
were carefully compensated (85–95%) before each voltage clamp
protocol run. The action potentials (APs) and firing were recorded in
current-clamp by means of a particular patch-clamp amplifier developed
in our laboratory (36)
or an Axopatch 200A in
Ifast mode (Axon Instruments, Foster City,
Calif.). The extracellular solutions were delivered through a 9-hole
(0.6 mm), remote-controlled linear positioner placed near the cell
under study, which has an average response time of 2–3 s, as shown in
Fig. 2a
(point line obtained after changing solutions at
different [K+]o). During
data acquisition and analysis, pClamp (Axon Instruments) and Origin 4.1
(Microcal, Inc., Northampton, Mass.) software were routinely used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a shows the chromatographic separation of ergtoxin, and Fig. 1b
the amino acid sequence with the overlapping fragments
used for the determination of the complete primary structure. The mass
spectrometry results showed a molecular mass of 4759 (data not shown),
which is within the range of the calculated molecular weight (MW) of
4757.3. As shown in Fig. 1c
, ErgTx is different from all of
the other known scorpion toxins affecting K+
channels. If we compare its primary structure with the 10 subfamilies
of other known scorpion toxins (7,
29,
30)
and add gaps (-) to increase
similarities, only cysteines (shown in boldface) overlap at equivalent
positions 8, 14, 23, and 42. The tripeptide Lys-Cys-Lys at positions
41–43 is also present in positions similar to those of the toxins
belonging to families 2, 4, 7, and 10 (underlined in Fig. 1c
). The pair of cysteines at positions 23 and 24 of the
primary structure (Fig. 1b
), corresponding to 26 and 27 in
Fig. 1c
, have no counterpart in any of the other toxins
known to affect K+ channels, including peptides
extracted from snake venoms (such as the dendrotoxin family), sea
anemone (such as BgK and ShK), and apamin from bee venom; in the spider
venom toxins hanatoxin 1 and 2, it is present in position 15–16 (for
review, see ref 7
). This means that ErgTx is a completely novel peptide
and may explain why it does not affect other known
K+ channels, as demonstrated below.
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Reversibility, speed, and dose-response relationship
IERG (in
[K+]o = 40 mM, see inset
to Fig. 2
a) was measured (20,
13)
in F-11 neuroblastoma cells using
the protocol shown, which consists of a series (0.25 Hz) of
depolarizations, followed by a brief test pulse necessary to evaluate
the number of open channels (as the inward tail current elicited at
-120mV). Figure 2a
shows the percent of the maximum
blocking effect over time obtained in an example cell (all cells
responded to the toxin). In comparison with a classical antiarrhythmic
blocker such as WAY-123398 (20,
25)
, which has a use-dependent block
(open triangles, 90% effect in 60 s), the toxin affected
IERG (open circles) in a rapid manner
(~10–12 s to reach 90% effect, see inset) that was hardly
distinguishable from the response time of the perfusing system (point
line). The recovery time constant was 33 ± 5 s at 500 ng/ml,
which is ~30 times faster than that of WAY. The concentration
dependence of the inhibition is plotted in Fig. 2b
. The data
have been fitted by the following equation: % unblocked channels =
(1+([ErgTx]/IC50)p)-1,
with an IC50 of 76 ± 2 ng/ml
(n=8) and P = 0.92, suggesting a putative
1:1 binding between ErgTx and the ERG channel, with an equilibrium
constant of 16 nM (given the MW of 4757.3).
Voltage dependence of the blocking mechanism in neuronal cells
To investigate its mechanism of blockade, we performed a series of
experiments to verify whether the peptide affected the
activation-inactivation gating of the channel. Figure 3
a shows the superimposed traces of the removal of
inactivation and subsequent deactivation of
IERG, in the range -20/-160 mV
compared with the scaled traces
(IERG/0.55) obtained in the presence
of the toxin at 100 ng/ml. No effects were evident, and specific
protocols designed to discover whether the voltage-dependent,
steady-state activation curve (panel b) was modified
revealed very small effects that were not further investigated. We
decided to check whether long-lasting conditioning at various holding
potentials (VH) could alter the inhibiting power
of ErgTx. From VH = +30 mV, we applied the
protocol shown in panel c (bottom) and tested the number of
open channels after having preconditioned the cell for a fixed time
(tPRE=3.5 s) at various levels
(VPRE). The results of this experiment at two
different drug concentrations (1 and 0.1 µg/ml) suggest an
interesting voltage-dependent block that can be fitted using Boltzmann
relationships with V1/2 values of (respectively)
-50.9 ± 1.6 and -24.2 ± 1.5 mV, and a slope factor of
22 ± 1.6 (n=3). The results of similar experiments
designed to investigate whether various tPRE
values influence the blocking at a constant VPRE
are shown in Fig. 3d
. They suggest that the toxin block is
also dependent on the time spent by the channels during their
deactivation at -70 mV (after the 30 ms removal of inactivation; see
panel a). From positive values of VH
(+30 mV), at which the channels are fully inactivated, ErgTx at 0.1
µg/ml is virtually unable to block channels. Blockade takes place
with a time constant of 7.6 ± 2.5 s (n=3), which
is the same as the deactivation time constant of the ERG channels in
these cells (20)
. At 1 µg/ml, ErgTx blockade from the inactivated
state has a time constant of 1.95 ± 0.1 s (n=3).
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On the whole, these experiments suggest that fully inactivated ERG
channels are not inclined to be blocked. This is substantiated by the
results shown in panel c, in which the voltage dependence of
the blockade resembles the inactivation curve of the ERG channels (20)
.
This type of blockade is different from what is known about the class
III antiarrhythmics, in which drug binding is dependent on the open
and/or inactivated state (27,
28)
. Although it is known that the
open-inactivated kinetics of ERG is relatively fast, the results shown
in panel d indicate that several seconds at -70 mV (8–10
s) are necessary to obtain the maximum block. This result, which
deserves further investigation, suggests that the channel must probably
be closed before being blocked by the peptide.
Ergtoxin also blocks cardiac and endocrine ERG channels
The current carried by erg-encoded channels in
cardiac myocytes is commonly referred to as IKr
(16,
18,
19)
. Figure 4
(panels a, b) shows the effect of 200 ng/ml ErgTx
on IKr in guinea pig ventricular cells. The toxin
effect on the total current activated by short depolarizing pulses is
shown in panel a. On average, current tails, mainly
contributed by IKr, were reversibly reduced from
16.5 ± 1.6 to 8.1 ± 1.1 pA by the toxin (n=9;
P<0.05) and from 16.5 ± 1.6 to 2.1 ± 0.6 pA by
the specific IKr blocker E-4031 (5 µM)
(n=12; P<0.05). Panel b shows the
difference current recorded during ErgTx superfusion
(IErgTx) (upper trace);
IErgTx was completely abolished by the specific
IKr blocker E-4031 (lower trace). Steady-state
activation curves for IKr (Fig. 4c, d)
, show that 200 ng/ml of toxin blocked ~50% of
IKr (panel c) and shifted its voltage
dependence (normalized curves in panel d) toward negative
potentials (
V1/2 = –8.4±4 mV; n=5;
P=0.05).
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A peculiar ERG-like current resembling the inward rectifier
currents of native pituitary cells (37)
is present in the MMQ (38)
dopamine-releasing rat clone expressing erg1,
erg2, and erg3 genes (10
; J. Schwarz, personal
communication). This is characterized by a long-lasting component of
deactivation as well as the fast deactivating component (39,
40)
, as
shown in the recordings of panel e elicited using the
protocol shown below. It can be seen that the fast deactivating
component has a threshold of around -60 mV, whereas the long-lasting
component has a threshold of between -120 and -60 mV. The currents
are almost completely blocked by 1 µM WAY, as shown in panel
f. The effect of ErgTx on this particular ERG current is
shown in Fig. 4g
, where it can be seen that ErgTx blocks the
WAY-sensitive currents.
We also tested the toxin on the series of tumor cells (13)
that
intrinsically express IERG; in all of
these cells we obtained a specific block that is very similar to that
studied in detail in the F-11 clone. Among these, the rat insulinoma
RINm5F endocrine cell line, which is known to express
Na+ and Ca2+ channels (41,
42)
, was used to test the action of the toxin on
IERG (which was blocked with an
IC50, similar to that found in neuronal cells,
not shown). These results suggest that ErgTx is active on a variety of
ERG channels expressed in different tissues of different species
(guinea pig, mouse, rat, and human).
Specificity of ergtoxin
To demonstrate the selectivity of the toxin, we performed
a series of experiments designed to screen a variety of other
K+ channels such as the classical inwardly
rectifying current (43)
(IIRK1), the
slow delayed rectifier IKs current,
the weak inwardly rectifying IK(ATP),
and the delayed rectifier current
IEAG.
A cell of muscular origin, such as the C2C12 clone, was used to test
the action of ErgTx (n=6) on IRK1 channels (44)
. An
exemplary inwardly rectifying K+ current
(elicited with the protocol of Fig. 5
c bottom, [K+]o
= 40 mM) is shown in Fig. 5
, panels a (control) and
b (in the presence of 1 µg/ml ErgTx). The application of
Ba2+ completely blocked the inward currents
(panel c). We therefore concluded that ErgTx does not
influence strong inward rectifier channels.
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It is known that IKs currents are
composed of heteromultimers of KVLQT and minK
(45,
46)
. Furthermore, it has been suggested that minK may form
heteromultimers with HERG proteins in CHO-transfected cells (47)
. Thus,
if ErgTx interacted with minK, IKs might also be
blocked by the toxin. This hypothesis was tested by measuring
IKs in guinea pig ventricular cells
(Fig. 5d-f
). As shown in Figure 5d
, IKs appeared to be slightly reduced during
superfusion with 400 ng/ml of ErgTx; however, this change was never
reversed by washout (not shown) and may have been due to spontaneous
current rundown (toxin effects in IKr were
quickly and fully reversible). In the same cell, the outward
time-dependent current was strongly inhibited by the specific
IKs blocker chromanol 293B (10 µM) (48)
(panel
e); the currents sensitive to chromanol
(Ichrom) and ErgTx (IErgTx)
obtained by trace subtraction are compared in panel f. Even
at 400 ng/ml of toxin, IErgTx was only 12 ±
2% (n=9) of Ichrom. The small
magnitude and the lack of reversibility of the
IKs changes suggest that this current is not
sensitive to ErgTx blockade.
The action of ergtoxin on K(ATP) channels was verified in human
pancreatic ß cells, which are known to express this type of channel
(49)
. By using the protocol shown in Fig. 5i
, we elicited
outward and inward currents in
[K+]o = 40 mM. Panels
g–i show the results of one of ten experiments in which the
currents were tested in control, ErgTx (1 µg/ml), and 50 µM
tolbutamide, respectively. It is obvious that ErgTx is not affecting
the current component blocked by tolbutamide, the specific
IK(ATP) blocker.
Finally, we investigated the possibility that ErgTx could block the EAG
channel belonging to the same superfamily, as ELK (9)
. In human
neuroblastoma SHSY5Y cells, EAG channels (50)
as well as HERG channels
(13)
are expressed. Figure 5j-k
shows the characterization
of IEAG. The recordings in panel
j show that, after patch breakdown, the current is
increasing during the first 4–5 min of the diffusion of a
Ca2+-free cytosolic solution into the cell (50,
51)
. Further evidence that the recorded current is
IEAG is given in panel k.
It is known (31,
50)
that IEAG is
characterized by a strong Cole-Moore effect on the rising phase when
elicited from strongly negative VH, and the
typical sigmoidal time-dependent current increase (label closed square,
elicited from -120 mV) is compared with a normal exponential time
course (open square, -60). There was no effect after the application
of ErgTx at a concentration (1 µg/ml, n=8), which is more
than 10-fold that of the IC50 for ERG channels.
Effects of ergtoxin on the excitability of neuronal and cardiac
cells
Recordings of firing and APs in current-clamp from excitable cells
were used to explore the physiological effects of the peptide and
confirm that other ion channels are not ErgTx targets.
It has been reported that the activation of ERG channels sustains
spike frequency accommodation in the neuronal firing of retinoic
acid-differentiated neuroblastoma F-11 cells (21)
. Figure 6
a–d shows that a typical pattern of spike frequency
accommodation (a) in a representative F-11 cell (of five
other cells) was transformed into a regular firing pattern after ErgTx
perfusion (b); after washout (c), the same effect
was reproduced with the specific blocker WAY (d). This
demonstrates that ErgTx has the same specificity as WAY. To show that
none of the other ion channels (33)
functioning during firing were
affected by ErgTx, we superimposed the APs of the control, ErgTx, and
WAY traces in the inset to panel d. The perfect overlapping
illustrates that neither the AP waveform nor the resting potential is
affected by ErgTx (panel b).
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The effect of the toxin on cardiac repolarization was tested in
ventricular myocytes stimulated at a rate of 1–2 Hz (Fig. 6e-f
). The values of the action potential duration
(APD90) measured from consecutive beats in a
representative cell are shown in panel e. On average, ErgTx
(200 ng/ml) increased APD90 from 148 ± 10
to 174 ± 12 ms (n=11; P<0.05), whereas
IKr blockade by 5 µM E-4031 prolonged
APD90 from 148 ± 9 to 185 ± 12 ms
(n=14; P<0.05). The complex pattern of the heart
cell APs was affected by ErgTx only at the level of repolarization that
is known (19)
to be controlled by the
IKr and
IKs, but Fig. 5
shows that the toxin
did not affect IKs tested in isolation
and establishes the specific nature of ErgTx action.
On the whole, these experiments show a direct action on nerve and cardiac cell excitability, and also indirectly reveal that Na+, Ca2+, and other K+ currents known to rule the excitability of F-11 and ventricular cells are not affected by ErgTx.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(scorpion, snake,
bee, or spider).
In this study, we identified and investigated a new member of the
scorpion venom family of K+ channel inhibitors.
These toxins fall into at least 10 distinct subfamilies whose relative
similarity is suggested by the alignment shown in Fig. 1c
.
It is known that there is also a significant overlap in specificity, as
members from the first three groups block Kv1.3
with high affinity (4,
7)
. From the comparison shown in Fig. 1c
, we discovered that ErgTx has a novel structure that
differs from those previously known, almost as if the toxin was
isolated from the venom of a new animal. The DCCK group at
position 25–28 of ErgTx is also present in the hanatoxins (position
14–17) from Grammostola spatulata spider venom (52)
(which
are specific for Kv2.1) and in heteropodatoxins
from the spider venom of Heteropoda venatoria, which is
specific for Kv4.2 K+
channels (53)
but not for EAG and ERG channels (52,
53)
.
Structure–function studies performed on the P region suggest that it
represents the only part of the channel that makes direct contact with
the first three scorpion toxins shown in Fig. 1c
(54)
. On
the contrary, regions outside the
S5-S6 linker have been
shown to determine the binding site to hanatoxins (52)
. What do these
separate toxin groups, defined on the basis of amino acid sequences,
mean in terms of channel specificity? A more direct connection between
function (channel inhibition) and the toxin groups defined above will
perhaps become apparent after more information on channel inhibition is
obtained.
Ergtoxin does not touch other K+ channels or, more important, the EAG channels belonging to the same eag superfamily as ERG. These results open up the possibility of investigating ERG channel structure–function relationships in detail and finding new pharmacological tools with potential therapeutic efficacy. The toxin search protocols described here could represent a key approach for finding specific toxins to the parent channels EAG and ELK, for which there are no selective blockers, making it difficult to isolate their role in cell physiology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations: ABS, adult bovine serum; AP,
action potential; DMEM, Dulbecco's modified Eagle
medium; erg, ether-a-go-go-related genes;
ERG, K+ channels; ErgTx, ergtoxin; FCS, fetal calf serum;
HPLC, high-performance liquid chromatography; IERG,
K+ channel current; IIRK1,
inwardly rectifying current; MW, molecular weight.
Received for publication October 30, 1998.
Revision received December 4, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. Diochot, E. Loret, T. Bruhn, L. Beress, and M. Lazdunski APETx1, a New Toxin from the Sea Anemone Anthopleura elegantissima, Blocks Voltage-Gated Human Ether-a-go-go-Related Gene Potassium Channels Mol. Pharmacol., July 1, 2003; 64(1): 59 - 69. [Abstract] [Full Text] [PDF]
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M. Zhang, Y. V. Korolkova, J. Liu, M. Jiang, E. V. Grishin, and G.-N. Tseng BeKm-1 Is a HERG-Specific Toxin that Shares the Structure with ChTx but the Mechanism of Action with ErgTx1 Biophys. J., May 1, 2003; 84(5): 3022 - 3036. [Abstract] [Full Text] [PDF]
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L. Pardo-Lopez, M. Zhang, J. Liu, M. Jiang, L. D. Possani, and G.-N. Tseng Mapping the Binding Site of a Human ether-a-go-go-related Gene-specific Peptide Toxin (ErgTx) to the Channel's Outer Vestibule J. Biol. Chem., May 3, 2002; 277(19): 16403 - 16411. [Abstract] [Full Text] [PDF]
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).
A protease V8 peptide corresponded to positions Cys20 to
Cys39 (labeled with [ - - - V8
