From the Research Institute of Pharmaceutical
Chemistry, Beijing 102205, China, the
Institute of Neuroscience,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China, the § Shanghai Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China, and the
** Laboratory of Toxicology, University of Leuven,
Leuven, Belgium
Received for publication, October 4, 2002, and in revised form, January 13, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A novel conotoxin, Biotoxins have been used widely to identify different
subtypes of ion channels in excitable cells. In many cases,
determination of a new subtype of ion channel has depended on finding a
specific biotoxin (1, 2). In particular, K+ channels are
much more diverse than Na+ and Ca2+ channels.
Therefore, new, subtype-specific biotoxins provide valuable tools to
identify distinct subtypes of K+ channels and to study
their functions in native systems (3-6).
Cone snails are predatory, venomous mollusks that use a common general
strategy to capture their prey. As a genus, cones use a rather diverse
spectrum of prey, including at least three different types: fishes,
other mollusks, and marine worms. Their venoms contain a large number
of small, conformationally constrained peptides that display highly
potent and specific biological activity. For example, A number of conotoxins were characterized in a recently published work
(15). In this study, we investigated the venom of a vermivorous
species, Conus betulinus, which is found in the South China
Sea. Although not highly toxic to vertebrates, the venom of C. betulinus causes obvious symptoms such as aggressiveness, stiff
tail, paralysis, convulsions, and even death when injected intraventricularly into mice. In the first part of this report, we
describe the purification and characterization of a new family of
conotoxins, In the second part of this report, we describe the electrophysiological
findings from whole cell patch clamp recording that Materials--
Sephadex G-25 was purchased from Amersham
Biosciences. Trifluoroacetic acid and acetonitrile for HPLC were from
Merck. The 3'-RACE and 5'-RACE kits, TRIzol reagent, and T4 DNA ligase
were purchased from Invitrogen. Restriction endonucleases and
TaqDNA polymerase were from MBI Company and Sangon Company,
respectively. The DNA sequencing kit was purchased from Promega, and
[ Peptide Purification--
Specimens of C. betulinus
were collected from Sanya, Hainan Province in the South China Sea. The
venom apparatus was dissected out, and the venom duct was cut in
sections and homogenized. The venom was then extracted with 1.1% (v/v)
acetic acid containing the protease inhibitor phenylmethylsulfonyl
fluoride. The sample was centrifuged at 12,000 rpm at 4 °C for 5 min. The residue was resuspended and soaked in the same buffer for 10 min, and then centrifuged again. Finally, the supernatants were
combined, lyophilized, and stored at Amino Acid Sequence Determination--
About 200 µg of the
purified peptide was dissolved in 1 M Tris-HCl, 6 M guanidine hydrochloride, and 1 mM EDTA at pH
8.5 and reduced with 2 mg of dithiothreitol at 37 °C for 5 h.
Then it was alkylated with 5 mg of idoacetic acid and kept in the dark for 30 min at room temperature. The modified peptide was purified by
HPLC. This carboxymethylated peptide (Rcm- Enzyme Hydrolysis--
Rcm- Mass Spectrometry--
Mass spectral analyses of the native
toxin, its modified form Rcm- 3'- and 5'-RACE--
3'- and 5'-RACE were used to clone the
cDNA of
5'-RACE is based on the partial cDNA sequence determined by
3'-RACE. A gene-specific primer 2 with a BamHI restriction
site (as underlined in 5'-CGGGATCCTACGACGAGAAGCA-3')
corresponding to the cDNA sequence downstream of the stop codon at
bp 21-34 was designed and synthesized. The first strand cDNA was
transcripted with primer 2 using 1 µg of total RNA as template. After
the cDNA was purified on a Glassmax column, the homopolymeric dC
was then tailed to its 3'-end. The dC-tailed cDNA was further
amplified using a nested primer 3 with an XbaI restriction
site 3 bp downstream from the stop codon (as underlined in
5'-CGTCTAGACAGACCTCTGAGCAAC-3') and an abridged anchor
primer (5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIG-3') complementary to the dC
tail. To obtain a high yield of toxin-specific cDNA, a second PCR
was performed using primer 3 and another anchor primer devoid of dG
with a HindIII restriction site (as underlined in
5'-GCAAGCTTACGCGTCGACTAGTAC-3'). The final PCR products
were sequenced as described above. The whole cDNA sequence of
Amplification of the Genomic DNA--
The total genomic DNA was
isolated from cone venom glands using a NaClO4 extraction
procedure followed by RNase treatment (16). Primer 4 with an
XbaI restriction site (as underlined in
5'-CGTCTAGA TGC CGC GCT GAA GGA-3'), corresponding to amino acid residues 1-5 (Cys-Arg-Ala-Glu-Gly), and primer 2 were used as a
pair to amplify the genomic DNA of Cell Culture--
RACCs were prepared from adult Wistar rats
(250-300 g) as described previously (17, 18). Single cells were
obtained after a 40-min digestion in enzyme solution. The cells
were cultured with Dulbecco's modified Eagle medium in a
CO2 incubator. Cells were used in experiments after 1-6
days in culture.
Electrophysiology--
The standard external solution
(bath solution) contained 140 mM NaCl, 2.8 mM
KCl, 2 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, at pH 7.4. The "high TEA
(tetraethylammonium chloride)" solution was the same as the standard
solution, except that the 20 mM NaCl was replaced by 20 mM TEA. The "high Cs+" solution was the
same as the standard, but KCl was replaced by CsCl. The standard
internal solution in the patch pipette contained 145 mM
KCl, 8 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 250 µg/ml nystatin, pH 7.2. Samples of
The voltage-gated membrane currents were recorded under whole cell
voltage clamp using the nystatin perforated patch clamp technique (3,
4). The series resistance (Rs) was typically
10-20 megaohms before Rs compensation. Before cell
recording, 75-90% Rs compensation was applied to allow fast voltage clamp of the voltage-dependent big
conductance K(Ca2+) channel (BK) currents. An outside-out
patch was obtained by excising the patch from a cell in the whole cell
configuration. Experiments were done using patch clamp amplifiers Axon
200B (Axon Instruments, Foster City, CA) and PC-2B (INBIO Inc., Wuhan,
China). Data were analyzed with Igor software (AveMatrix, Lack
Oswego, OR).
The Data Analysis--
Each data point of a dose-response curve
represents the mean ± S.D. of at least five cells.
Dose-response curves were fit to Equation 1
To define the average open probability
(Po) of single channels in a patch containing
more than one BK channel, we determined the probability that
r channels were open simultaneously (19). The parameter
r follows a binomial distribution, as shown in Equation 2
Amino Acid- and cDNA-deduced Sequences of Conotoxin
The deduced amino acid sequence consisted of a putative signal peptide
of 26 residues and a protoxin of 44 residues. According to the amino
acid analysis (data not shown) and the partial N-terminal sequence, the
sequence of the mature toxin devoid of an extra C-terminal was as
follows: CRAEGTYCENDSQCCLNECCWGGCGHPCRHP* (the asterisk
indicates C-terminal amidation). The C terminus of the mature
toxin was followed by an extra 13-residue peptide, as a propeptide,
which was removed during post-translational processing. The calculated
molecular mass of this mature toxin was 148 Da less than that measured.
Furthermore, there was a 132 Da difference between the LCQ-MS and the
MALDI-TOF-MS analyses (Table I). These differences suggested that the toxin might be post-translationally modified, for example by carboxylation of glutamate, hydroxylation of
proline, and amidation of the C terminus, as is often the case for conotoxins. If all three Glu residues at positions 4, 9, and 18 were carboxylated (Gla), one of the two Pro residues at position 27 or
at 31 hydroxylated, and the C terminus amidated, then the calculated
molecular weight (3569.2) would be quite consistent with the measured
value (3569.0). Furthermore, the presence of Gla residues was verified
by the mass spectrometric analysis. When the MALDI source was used
instead of the electrospray ionization source, the decarboxylation of
the peptide resulted in a total loss of 132 Da, corresponding to three
decarboxylated groups of Gla residues.
To determine in which position the hydroxyproline was located, the
reduced and carboxymethylated conotoxin was further digested with
TPCK-trypsin. As there are only two cleavage sites for TPCK-trypsin in
the toxin (Arg at positions 2 and 29), the enzymatic cleavage removed
two peptides, both with two residues, from the N and C termini. The
remaining fragment (from residues 3 to 29) was then purified on HPLC
and subjected to mass spectrometric analysis. A mass of 3492 Da was
measured, matching quite well with the calculated value (3492.5 Da).
Thus, the hydroxyproline was located at position 27, and the full
sequence of Cloning and Sequencing Genomic DNA of Effects of Up-modulation of BK Currents by Selectivity of
There is controversy in the literature concerning the effects of
charybdotoxin (ChTX) on SK channel, which is another type of
Ca2+ dependent K channel in RACCs (5). To compare the
effects of
That the BK channels were the targets of
The time course of onset and recovery of the up-modulation effect was
relatively fast in comparison with other biotoxins. The time constant
of the Mechanisms of
10 nM This report describes the purification, characterization, and mode
of action of a novel Conus peptide, Structure of
Among the known precursor sequences of the Conus
peptides, the mature peptides are usually present in the C-terminal
part in a form of pre-propeptide. However, the pre-propeptide sequence of Specificity of
The scorpion toxin ChTX is a well known BK blocker and has been widely
used in BK studies (2). ChTX is considered to be selective only when
one compares K+ with Na+ and Ca2+
channels. ChTX is not selective between the subtypes of K+
channels because it blocks not only BK but also SK channels (Fig. 6,
C and D) and delayed rectifier K+
channels (2). The other scorpion toxin, IbTX, is a specific blocker for
BK channels versus other delayed rectifier K+
channels (5). In this work, Up-modulator of BK Channels--
So far, five compound types have
been reported to activate the BK channel: a medical herb,
anti-inflammatory aromatic compounds, benzimidazolones, phloretin, and
ethanol (28). However, none of them are selective, and they are less
potent than
As an up-modulator of BK channels, the biological role of Mechanisms of
The binding site between ChTX and the BK channel is located in the
pore-forming region (31). At present, it is not clear where the exact
location of the binding site between
Although more than 80 conotoxins have been characterized, only 2 of
them, -conotoxin (
-BtX),
has been purified and characterized from the venom of a worm-hunting
cone snail, Conus betulinus. The toxin, with four disulfide
bonds, shares no sequence homology with any other conotoxins. Based on
a partial amino acid sequence, its cDNA was cloned and sequenced.
The deduced sequence consists of a 26-residue putative signal peptide,
a 31-residue mature toxin, and a 13-residue extra peptide at the
C terminus. The extra peptide is cleaved off by proteinase
post-processing. All three Glu residues are
-carboxylated, one of
the two Pro residues is hydroxylated at position 27, and its C-terminal
residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels,
-BtX is a specific modulator of K+ channels;
2) among the K channels,
-BtX specifically up-modulates the
Ca2+- and voltage-sensitive BK channels (252 ± 47%);
3) its EC50 is 0.7 nM with a single binding
site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5)
-BtX has no effect on single channel conductance, but
increases the open probability of BK channels. It is concluded that
-BtX is a novel specific biotoxin against BK channels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxins
are competitive antagonists of the nicotinic acetylcholine receptor;
- and µ-conotoxins act at sites VI and I of voltage-sensitive
sodium channels, respectively;
-conotoxins selectively inhibit
presynaptic calcium channels at neuromuscular junctions; and
-conotoxin PVIIA inhibits Shaker-type potassium channels
(7-13). The conotoxins are much smaller than other peptide toxins,
with only 10-30 amino acids. The pattern of disulfide bridges of each
conotoxin family is relatively conserved (14). A large number of
conotoxins have been characterized, most of them from piscivorous
(fish-hunting) and molluscivorous (mollusk-hunting) snails, since they
have relatively high toxicity to vertebrates. In contrast, the venom of
vermivorous (worm-hunting) snails has been seldom studied.
-conotoxin BtX
(
-BtX),1 by using protein
sequence determination, gene cloning, and functional assays. This toxin
is characterized by the presence of four instead of two or three
disulfide bonds, as usually found in other conotoxins, and shares no
sequence homology with any others.
-BtX is a
K+ channel up-modulator, not a blocker, targeting the
Ca2+ and voltage dependent K+ channel in rat
adrenal chromaffin cells (RACCs).
-BTX is therefore a novel and
unique pharmacological tool to dissect out the functional properties of
the different subtypes of K+ channels.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP was purchased from Amersham Biosciences.
Acrylamide, bisacrylamide, 5-bromo-4-chloro-3-indolyl-
-D-galactoside (X-gal),
isopropyl-thio-
-D-galactoside, and all other reagents
were of analytical grade. All chemicals used in the
electrophysiological experiments were from Sigma.
20 °C. Lyophilized samples
were reextracted with 1.1% acetic acid. After centrifugation, the
supernatant was placed on a pre-equilibrated Sephadex G-25SF column
(100 × 2.6 cm) and eluted with 1.1% acidic acid at 4 °C. The
fractions were collected at flow rate of 0.35 ml/min (Fig.
1A). The fractions from each peak were pooled and lyophilized. The freeze-dried fractions IV from
gel filtration were dissolved in 0.1% trifluoroacetic acid and applied
to HPLC with a Phenomenex C18 semipreparative column (25 × 1 cm,
micron). The peptides were eluted with a gradient of 0.1%
trifluoroacetic acid in acetonitrile. The
-BtX fraction was located
in peak 5 (Fig. 1B), which was repurified under the same
conditions.
View larger version (16K):
[in a new window]
Fig. 1.
Purification of
-conotoxin BtX. As shown in A,
extracted crude venom was applied to a Sephadex G-25SF column (100 × 2.6 cm) eluted with 1.1% acetic acid. The fractions were collected
at a flow rate of 0.35 ml/min. As shown in B, freeze-dried
fraction IV was applied to a Phenomenex C18 semipreparative column
(25 × 1 cm) and eluted with a linear gradient from 100% A, 0% B
to 55% A, 45% B over 45 min and then with 100% A, 0% B for 5 min at
a flow rate of 2 ml/min. Buffer A was 0.1% trifluoroacetic acid;
buffer B was 0.1% trifluoroacetic acid in acetonitrile. The
-BtX
fraction was present in peak 5, which was repurified under the same
conditions.
-BtX) was directly used
for automatic sequencing on a PE ABI model 491 peptide/protein sequencer and PTH analyzer using the program provided by the manufacturer.
-BtX- and TPCK-treated trypsin
were mixed at a ratio of 50:1 and incubated in 0.5 ml of buffer (20 mM Tris-HCl, 10 mM calcium chloride, pH 7.8) at
37 °C for 10 h. The cleaved fragments were separated and
purified on a C-18 reverse-phase HPLC.
-BtX, and degraded fragments were
performed on a Finnigan LCQ ion trap mass spectrometer (ThermoQuest,
San Jose, CA). The apparatus was equipped with an electrospray
ionization source with a spray voltage of 4.5 kV. Mass spectra of the
native toxin were also obtained using a Q-TOF mass spectrometer
(Micromass, Manchester, UK) equipped with an electrospray ionization
source and a matrix assistant laser desorption ionization (MALDI) TOF mass spectrometer (Brucker Biflex III).
-BtX from the fresh venom ducts of C. betulinus by using the TRIzol reagent kit. Five µg of total RNA
was converted into cDNA using superscript II reverse transcriptase
and a universal oligo(dT)-containing adapter primer
(5'-GGCCACGCGTCGACTAGTAC(dT)17-3'). Using the degenerate codons, a gene-specific primer 1 with a BamHI
restriction site (as underlined in 5'-CGGGATCC GCC AAC GGT
AC(G/A/T/C) TA(T/C) TG-3') encoding residues 3-8 of
-BtX was
designed and synthesized. Primer 1 was paired with an abridged
universal primer with a HindIII restriction site (as
underlined in 5'-GCAAGCTTACGCGTCGACTAGTAC-3') similar to
the adapter primer but devoid of the polyT for 3'RACE amplification.
The amplification products with a size of about 300 bp were purified,
cloned into the T-vector, and transformed into DH5
, and the positive
clones were sequenced.
-BtX was generated by combining the two fragments amplified by
3'- and 5'-RACE.
-BtX. The amplified product with
a size of around 200 bp was purified and cloned into the T-vector for sequencing.
-BtX were dissolved in the bath solution at the final concentration needed.
-BtX was applied to the cell under investigation through a glass
micropipette by applying slight positive pressure (3). Control/wash-out
solutions and toxins other than
-BtX were puffed locally onto the
cell via an RCP-2B multichannel microperfusion system (INBIO Inc.,
Wuhan, China), which allowed fast change of solutions by electronic
switching between seven solution channels (18). The puffer pipette
(100-µm tip diameter) was located about 120 µm from the cell. We
determined, by a conductance test using pure water as perfusion
solution, that the recorded cell would only be contacted by puffer
solution if the application speed was 100 µl/min or faster. All of
the pharmacological experiments in this study met this requirement. All
these experiments were done at room temperature (22-25 °C).
where
(Eq. 1)
is the relative response (%); n is the
Hill coefficient; [toxin] is the drug concentration; and
EC50 is the dissociation constant.
where n is the total number of channels in the patch
and Po is the open probability. Equation (2) was
used to fit the mean open time distribution and determine
Po. This formula is ideal for non-inactivating
channels. However, since the inactivation rate of BK channels was not
altered by
(Eq. 2)
-BtX (see Figs. 4 and 7), we could still use this formula
to calculate relative changes of Po
with and without
-BtX. Total open time of the single channels was
defined as the sum of open times from all single channels during a
given pulse. If two channels opened simultaneously, the total open time
was the sum of the open times of both channels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-BtX--
The
-BtX purified from C. betulinus venom
was subjected to automatic sequencing directly after reduction and
carboxymethylation. The partial N-terminal sequence was determined as
follows: CRANGTYCNNDSQCCLN. Since the retention
times of PTH-Gla and PTH-Asn were very close, Gla residues 4 and 9 were
at first misinterpreted as Asn (underlined). According to the
N-terminal partial sequence of the toxin, the gene-specific primer 1 was designed and synthesized for 3'-RACE. Despite the mismatch in the
synthesized oligonucleotide resulting from the misinterpreted amino
acid in position 4 (corresponding to codon AAC instead of GAA in the
primer), the 3'-RACE could still be performed successfully. Based on
the partial 3'-cDNA sequence, primer 2 was used for the 5'-RACE.
The whole cDNA sequence of the conotoxin
-BtX was subsequently
obtained by combining the two amplified fragments (Fig.
2).
View larger version (22K):
[in a new window]
Fig. 2.
cDNA and predicted amino acid sequence
of -conotoxin BtX.
Measured and calculated molecular masses for -BtX
-BtX is as follows: CRA
GTYC
NDSQCCLN
CCWGGCGHOCRHP* (
is
-carboxyglutamate; O is
hydroxyproline; the asterisk indicates C-terminal amidation).
-BtX--
Based on the
elucidated cDNA sequence of
-BtX, primers 2 and 4 were used to
amplify the genomic DNA corresponding to the
-BtX gene. The
analysis showed that the genomic sequence is identical to the cDNA
sequence. This finding implies that there is no intron in the genomic
DNA structure of
-BtX.
-BtX on Voltage-gated Channels in Chromaffin
Cells--
Chromaffin cells are endocrine cells that differentiate
from the neural crest during embryonic development. They are excitable cells with typical neuronal voltage-gated Na+,
K+, and Ca2+ channels and are widely used as a
neuronal model (3, 17, 22-24). To investigate whether
-BtX affects
voltage-gated ion channels, we studied the effect of
-BtX on these
channels in RACCs (Fig. 3). In Fig.
3A, the top panel shows current traces induced by
a voltage ramp (bottom panel). Three ramp-induced currents were superimposed. The cell was stimulated by the first ramp in the
absence of toxin (Control). The inward transient current was activated at about
30 mV and corresponded to a typical, voltage-gated Na+ current. The outward current (positive current
component) was activated from
30 mV and increased in amplitude toward
positive voltages, as expected for a typical, voltage-gated outward
K+ current (17, 24). The cell was subsequently stimulated
by a second ramp in the presence of 1 µM of
-BtX,
which resulted in a 2-fold increase of the peak outward current. The
up-modulating effect on the outward current was statistically
significant. The K+ current in response to the third
ramp, after removal of
-BtX, recovered to the control level. Along
with Na+ and K+ currents, RACCs have
voltage-gated Ca2+ channels of the L-, N-, P/Q-, and
R-types (23). Fig. 3B shows the effect of
-BtX on
Ca2+ currents, which were sensitive to 200 µM
Cd2+ (data not shown). To isolate the Ca2+
currents, the Na+ current was blocked by removing
Na+ from the external solution. All voltage-gated
K+ currents were blocked by high extracellular TEA and high
internal Cs+. The three Ca2+ current traces
were recorded as control, test (1 µM
-BtX), and recovery, sequentially at 120-s intervals. All three Ca2+
currents were nearly identical, indicating that
-BtX had little or
no effect on the voltage-gated Ca2+ channels
(n = 7).
View larger version (14K):
[in a new window]
Fig. 3.
Effects of -BtX on
Na+, K+, and Ca2+ currents in
RACCs. As shown in A, whole cell currents were induced
by 100-ms voltage ramps (
100 to 100 mV).
-BtX had negligible
effects on the downward (inward) Na+ current. However, it
increased the outward K+ current around 2-fold. The average
up-modulation was 164 ± 10% (p < 0.01, n = 11). B, effect of
-BtX on
Ca2+ currents. Ca2+ currents were induced by
depolarizing pulses in high TEA (20 mM) in the bath and
high CsCl (145 mM) internal solution. All records were from
the same cell with 90-s intervals. The Ca2+ current induced
during
-BtX application was nearly the same as in control. The
slight decline of Ca2+ current during the recovery was due
to the rundown of Ca2+ channels.
-BtX--
The target of
-BtX was found to be the BK channel (Fig.
4). In a standard double-pulse protocol
to test the BK current (25), the first step of the double pulse (from
70 to 0 mV for 100 ms) induced Ca2+ influx through the
voltage-gated Ca2+ channels, which activated the typical
large and fast-inactivating BK currents during the strong
depolarization of the second step (from 0 to 80 mV for 500ms, Fig.
4A, trace 1). Most BK currents were absent when
the prepulse for loading Ca2+ was omitted (Fig.
4A, trace 2). The difference between traces 1 and 2 yielded the pure BK currents, and this
difference was used to determine the effects of
-BtX (Fig.
4B). During application of 10 nM
-BtX, the BK
currents induced by the double-pulse protocol were 2.3 times greater
than the control (or the current after wash-out). On average,
-BtX
(10 nM) increased (or up-modulated) BK currents by
2.51 ± 0.47 times control (n = 65, Fig.
4C). Since BK current was the dominant component in the
double-pulse protocol, the "pure BK currents" in Fig. 4B
were very similar to trace 1 in panel A. The
dose-response curve of the up-modulation effect is illustrated in Fig.
4D.
-BtX had an EC50 of 0.7 nM
for BK channels. The Hill coefficient was 0.88, indicating that there was a single binding site between
-BtX and the BK channel.
View larger version (20K):
[in a new window]
Fig. 4.
-BtX increases
Ca2+- and voltage-dependent BK channel
currents. A, the protocol to record BK currents in
RACC. Two current traces from the same patch were recorded and
superimposed according to the stimulation time (upper). The
first voltage protocol (solid line) consisted of two pulses.
The second voltage protocol (dashed line) was similar to the
first protocol, except there was no prepulse before the 80 mV pulse.
The current induced by the second (no prepulse) protocol
(trace 2) was mainly due to Kv channels (see "Results").
The difference between traces 1 and 2 gave the
pure BK current (n > 100). As shown in B,
-BtX increased the pure BK current by 229% in this cell. The traces
show the pure BK currents obtained with the protocol described in
panel A. The three current traces, before, during, and after
application of 10 nM
-BtX, were superimposed according
to stimulation time. C, statistics of
-BtX up-modulation
of BK currents shown in panel B. On average,
-BtX
increased BK currents by 2.52 ± 0.47-fold (p < 0.01, n = 65). D, dose-response curve of
-BtX up-modulation of the pure BK currents. The pure BK current was
elicited by the protocol shown in panel A. The curve was
fitted by the Hill function (see "Experimental Procedures"). The
EC50 was 0.7 nM, and the Hill coefficient was
0.88 (n = 5).
-BtX among K+ Channels--
We found
that the BK current was the exclusive subtype of voltage-gated
K+ channel sensitive to
-BtX in RACCs (Fig.
5). Pure Ca2+-independent but
voltage-dependent K+ current, or Kz
current, can be recorded in Ca2+-free bathing solution. The
"Kz channel" is a delayed rectifier channel with fast
activation and slow inactivation kinetics (6, 24). In the presence of 2 mM Ca2+, application of 10 nM
-BtX increased K+ currents induced by the step
depolarization pulses 2-fold, having evoked both Ca2+
influx and BK currents. When the bath was changed to a
Ca2+-free solution,
-BtX failed to increase the
depolarization-induced K+ current. Statistically,
-BtX
increased total depolarization (single pulse)-induced currents by
67 ± 7% with 2 mM Ca2+ and
2 ± 5% in the absence of Ca2+ (Fig. 5B,
n = 5).
View larger version (17K):
[in a new window]
Fig. 5.
-BtX has no effect on
Ca2+-independent but voltage-dependent
K+ currents. As shown in A,
-BtX has no
effect on K+ currents in Ca2+-free bath. The
outward current of the depolarization-induced currents was
K+ current.
-BtX increased the outward K+
current to 160% of the control level in 2 mM
Ca2+ bath (top panel). However,
-BtX had no
effect on the outward K+ current in 0 Ca2+ bath
(middle panel), indicating that all
Ca2+-independent K+ currents were insensitive
to
-BtX. All traces were from the same cell. As shown in
B, on average,
-BtX increased the BK current to 167 ± 7% of control. In the same cells, removing Ca2+ from
the bath abolished the
-BtX effect completely (98 ± 4% of
control). This suggests that
-sensitive K+ currents are
exclusively Ca2+-dependent (n = 5).
-BtX and ChTX, we reexamined the effect of ChTX on SK
channels. Pure SK currents were examined by another type of
double-pulse protocol (Fig.
6A) (17, 26, 27). Again the
prepulse (from
70 mV to 0 mV for 1 s) induced Ca2+
influx, which activated a Ca2+-dependent and
voltage-independent inward current (thick trace) during the
hyperpolarization of the second pulse (from 0 mV to
100 mV). The
hyperpolarization deactivated all voltage-dependent currents, including the BK current. Most of the inward current at
100
mV was absent when the Ca2+-loading prepulse was omitted
(Fig. 6A, thin trace). This
Ca2+-sensitive inward current at
100 mV was assumed to be
an SK current because the specific SK antagonist apamin (100 nM) blocked it by 50% (Fig. 6B). In contrast to
the BK current, the extracellular application of a very high
concentration of
-BtX (1000 nM) induced only a minor
increase (~10%) in the SK current (Fig. 6B). In contrast, the BK channel antagonist ChTX (200 nM) blocked 53 ± 3% of SK currents, so it is as potent as the specific SK blocker
apamin (Fig. 6, C and D).
View larger version (21K):
[in a new window]
Fig. 6.
-BtX has little effect on SK
currents. A, protocol to detect pure SK in RACCs. The
slow tail current was induced by the double-pulse protocol and recorded
during the second pulse at
100 mV in 2 mM
Ca2+ bath solution. The SK current was activated by
Ca2+ influx during the 1-s prepulse in the 2 mM
Ca2+ bath (thicker trace). In the same cell, SK
currents were absent when no prepulse was given to induce
Ca2+ influx (thinner trace, n = 4). As shown in B, SK currents were induced using the
protocol described in panel A before, during, and after
application of
-BtX. In this cell,
-BtX (1000 nM)
increased the SK current by only 11%. The average increase of SK by
-BtX was 9 ± 4% (n = 13). Apamin (100 nM) blocked 50% of the current, indicating that the
Ca2+-sensitive inward current at
100 mV was indeed SK
current. C and D, effects of ChTX on SK channels.
SK was blocked by 200 nM ChTX (D). The
dose-response curve of ChTX shows that 200 nM blocked
52 ± 3%. The Hill coefficient was 0.95, indicating a single
binding site between ChTX and SK (n = 8).
-BtX was further supported
by experiments using the BK blocker ChTX. As shown in Fig.
7A, up-modulation of the BK
current by 10 nM
-BtX was largely removed by 100 nM ChTX. The
-BtX induced up-modulation was readily reversible; after a 5-min wash-out, the up-modulation recovered by 90%
(Fig. 7A). Since the Kd of
-BtX was
0.7 nM (Fig. 4C), 10 nM was a
saturating concentration and produced the maximum effect. Note that the
peak K+ current in the presence of ChTX +
-BtX was even
smaller than that without them. This was probably because without
-BtX, the basal BK current (Fig. 4A) was blocked even
more by ChTX (Fig. 7B). Before adding ChTX,
-BtX produced
a 68 ± 10% increase of the BK current (Fig. 7B,
n = 7). BK current can be blocked more effectively by 1 mM TEA (24). Indeed, 1 mM TEA blocked 85% of the total K current. After 1 mM TEA blockade of BK, the
effect of
-BtX on K current was abolished. Taken together, these
experiments provide further independent evidence that
-BtX is a
specific up-modulator of the BK channel.
View larger version (16K):
[in a new window]
Fig. 7.
ChTX blocks -BtX
induced BK up-modulation. As shown in A, ChTX (100 nM) blocked the
-BtX (10 nM)-induced BK
up-modulation. BK currents induced by the double-pulse protocol shown
in Fig. 1 were superimposed according to stimulus time. All traces were
from the same whole cell patch (n = 7). As shown in
B, data were measured using the protocol shown in
panel A. The up-modulation was normalized to BK current
under control conditions.
-BtX increased BK current to 168 ± 11% of control level. ChTX reduced the total K+ current to
30 ± 6% (without
-BtX, n = 6) and 71 ± 4% (with
-BtX, n = 7) of its control level
(p < 0.01). TEA (1 mM) reduced the
total K+ current to 15 ± 2% (without
-BtX,
n = 8) and 17 ± 3% (with
-BtX,
n = 3) of its control level.
-BtX wash-out curve was 8.3 s (Fig. 8, n = 7). The delay in
the puffer device was less than 0.1 s (not shown). The wash-out
time of
-BtX was much faster than that of ChTX (>3
min)2 and iberiotoxin (IbTX)
(5). The association rate was too fast to be determined by our
application system. (We did not determine the onset of the
-BtX action because the drug dialysis at the tip of the puffer
pipette prevented testing rapid onsets.)
View larger version (11K):
[in a new window]
Fig. 8.
Time course of -BtX
wash-out. The time constant of
-BtX (10 nM)
wash-out was 8 s, showing that the
-BtX effect on BK channels
was fast and completely reversible (n = 6). The
up-modulation curve increased to a maximum within about 10 s,
which was the typical time required for the puffer system to reach a
steady-state concentration of
-BtX.
-BtX Action on BK--
Next, we studied the
mechanism of
-BtX action on the BK channels via single channel
recordings in outside-out patches. As shown in Fig.
9,
-BtX had no effect on single BK
channel current (Fig. 9A) or single channel conductance
(Fig. 9B), suggesting that the up-modulation induced by
-BtX was not due to changes in single channel conductance. Note that
during
-BtX application, two or three single BK channels opened
simultaneously. In contrast, only a single channel current was visible
in the same patch without
-BtX.
View larger version (19K):
[in a new window]
Fig. 9.
-BtX has no effect on single
channel conductance of BK channels. A, typical sweeps
from an excised outside-out patch before and after 10 nM
-BtX applications at different depolarization potentials. The patch
was exposed to 2 µM Ca2+ in the pipette and
stepped every 3 s from
80 mV to 20 mV (top panel) or
to 80 mV (middle panel) for 500 ms.
-BtX had no effect on
the amplitude of single channel currents. The solutions for the
outside-out patch were asymmetric K+ solutions (see
"Experimental Procedures"). As shown in B,
-BtX had
no effect on the I-V curve of the single channel current. The single
channel conductance was 286 and 284 picosiemens for control and in the
presence of 10 nM
-BtX, respectively. C,
statistics of the effect of
-BtX on the single channel conductance.
The average conductance was 290 ± 13 picosiemens for control and
289 ± 12 picosiemens for
-BtX (n = 6).
-BtX increased the open probability and total
open-time of single BK channels in an outside-out patch (Fig.
10). Single channel BK currents were
induced by depolarization from
80 mV to 80 mV with 2 µM
Ca2+ in the patch pipette in the presence or absence of
-BtX (Fig. 10A). Statistical analysis revealed that
-BtX increased the open probability by 2.45-fold and total channel
open time (see "Experimental Procedures" for definition) by
1.93-fold in single BK channel recordings (p < 0.01, Fig. 10, B and C). These values were similar to
those of the whole cell BK currents (Figs. 4, 5 and 7). Taken together,
the mechanism of
-BtX induced up-modulation of BK currents is that
-BtX increases the open probability and thus total open time of
single BK channels during a given sweep but does not affect the single
channel conductance.
View larger version (34K):
[in a new window]
Fig. 10.
-BtX increases open
probability of single BK channels. A, examples of
sweeps from an excised outside-out patch before and after 10 nM
-BtX applications. The patch was exposed to 2 µM Ca2+ in the pipette and stepped every
3 s from
80 mV to 80 mV for 500 ms. B, the effect of
10 nM
-BtX on the total open time of single BK channels.
After application of 10 nM
-BtX, the total open time
increased to 1.94 ± 0.48-fold over the control. The effect of 10 nM
-BtX on the total open time was statistically
significant (p < 0.01, n = 6).
C, the effect of 10 nM
-BtX on the open
probability of single BK channels. After application of 10 nM
-BtX, the open probability increased to 2.45 ± 0.5-fold over the control. The effect of 10 nM
-BtX on
the open probability was statistic significant (p < 0.01, n = 6).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxin BtX. The peptide acts as a specific up-modulator of the Ca2+- and
voltage-dependent BK channel and does not act as a channel blocker as found for all other conotoxins. Its primary amino acid sequence, including post-translational modifications, was established by partial Edman degradation, 3'- and 5'-RACE amplification, mass spectrometric analysis, and digestion of the carboxymethylated toxin
with TPCK-trypsin. Our electrophysiological experiments showed that: 1)
-BtX had little effect on voltage-gated Na+ and
Ca2+ channels, but it increased voltage-gated
K+ currents; 2) the BK channel was sensitive to
-BtX,
with an average increase of 252 ± 47%; 3) other K+
channels, including the Ca2+-insensitive but
voltage-sensitive Kz channel and the
Ca2+-sensitive but voltage-insensitive SK channel, were not
sensitive to
-BtX; and 4)
-BtX affected the open probability but
not the conductance of single BK channels.
-BtX--
From the viewpoint of the topological
structure, the peptide is quite different from other known
Conus peptides. One unique feature is that
-BtX
contains 8 cysteine residues to form four intramolecular disulfide
bonds in a framework of
CX6CX5CCX3CCX3CX3CX3. Thus,
-BtX represents a new cysteine framework lacking sequence homology with any other conotoxins. Furthermore, the post-translational modifications of
-BtX are more complex than those of other
conotoxins, with all three Glu residues being
-carboxylated, one Pro
at position 27 hydroxylated, and another C-terminal Pro amidated. In
accordance with the proposed rules for conotoxin nomenclature (13), the Roman numeral in a toxin designation should represent its cysteine framework. As the Roman numerals I to IX have already been assigned, the numeral X is therefore designated for the proposed new cysteine framework of
-BtX (14).
-BtX is remarkably different. The deduced amino acid sequence consists of a putative signal peptide of 26 residues, a mature toxin of
31 residues and an extra tail of 13 residues at the C terminus. The
extra peptide might act as a propeptide and might be removed during
post-translational processing. It is well known that the residue Gly is
inevitably required for the C-terminal amidation of any protein by
peptidylglycine
-amidating monooxygenase. The presence of Gly was
also confirmed by the cDNA-deduced sequence of
-BtX.
Furthermore, this Gly residue is followed by two basic residues, Lys
and Arg, that might function as a recognition site for the amidation enzyme.
-BtX--
One important advantage of biotoxins
has been their specificity against a particular ion channel, such as
tetrodotoxin against the Na+ channel. However, it was
difficult to find biotoxins with high specificity for subtypes of
K+ channels, such as BK channels, other than the scorpion
toxins ChTX and IbTX.
-BtX was found to specifically up-modulate the BK current but not voltage-gated Na+ and
Ca2+ channels in RACCs.
-BtX has little effect on other
subtypes of K+ channels including Kz and SK
channels. To our knowledge,
-BtX is the first K+ channel
up-modulator from a Conus venom.
-BtX, except for DHS-I, an organic compound from
a medical herb (29). However, DHS-I increases the BK current only when
it is applied intracellularly. Since DHS-I is membrane impermeable, it
can be used only when intracellular dialysis with a
glass-pipette-electrode is available, which limits its wide
application. Thus,
-BtX is superior because it can affect BK
channels extracellularly.
-BtX is
opposite to that of BK blockers. It is known that the BK antagonists
ChTX and IbTX reduce the frequency of action potentials and broaden the
spike duration in neurons and adrenal chromaffin cells (8,
9),3 because BK channels are
required for fast repolarization between action potentials. In these
cells,
-BtX should increase action potential frequency and reduce
the spike duration. In other cells, the function of BK channels is to
keep cells at the resting potential. In these cells, BK up-modulators
would act similarly to blockers of voltage-gated Na+
channels (tetrodotoxin) or Ca2+ channels (nifedipine,
-conotoxin). The worm-hunting snail might use this latter function
as its weapon to catch prey, just like the
-conotoxin case (30).
-BtX Effects--
Single channel recording of BK
channels revealed that
-BtX had no effect on single channel
conductance (Fig. 9). Instead,
-BtX increased the open probability
of BK channels 2.45-fold. This is close to the 2.52-fold increase of
the BK whole cell current (Fig. 4). Thus, the mechanism of action of
-BtX is that the BK channel opens more frequently after binding with
it. There are two possibilities. First,
-BtX may increase the total
open time by increasing the Ca2+ sensitivity of the
channel. Alternatively,
-BtX may act directly on the gating site of
the channel. Since the Ca2+ sensor is intracellular, it is
more likely that
-BtX directly affects the channel gating when it is
opened by the combined action of Ca2+ and depolarization.
-BtX and BK channel is.
However, the present data provide some useful cues. Firstly, the
binding site might be different from that of ChTX because
-BtX did
not remove the ChTX block (Fig. 7). Secondly, BK channel inactivation
is through an intracellular "peptide ball" at the N-terminal of the
-subunits (20, 32), and the binding site of
-BtX is not likely to
be associated with
-subunits because the inactivation kinetics
remain intact in its presence (Fig. 3A). Finally, the single
binding site is probably located at the extracellular side of the
channel because the
-BtX effect can be washed out within seconds
(Fig. 8).
-conotoxin PVIIA and
A-conotoxin SIVA, target potassium
channels (17, 21). Both toxins inhibit voltage-gated potassium
channels. On the contrary, the present study has identified
-BtX as
an up-modulator of BK channels in chromaffin cells. Thus,
-BtX
provides a useful new pharmacological tool for the characterization of
K+ channels and possibly even for application in the
treatment of disorders caused by membrane hyper-excitability (21).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Martin Stocker (University College London, London, UK), Christopher Lingle (Washington University, St. Louis, MO), and Iain Bruce (Hong Kong University, Hong Kong) for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants 39525009, 39970238, and 39970371 from Natural Science Foundation of China, The Li Foundation (San Francisco), and Major State Basic Research Program of China (Grant G2000077800) (to Z. Z.) and Major State Basic Research Program of China (Grant G1998051121) (to C. W. C.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF208661.
¶ These authors contributed equally to this work.
Partially supported by `BIL 00/06'.
§§ To whom correspondence may be addressed: Research Institute of Pharmaceutical Chemistry, Beijing 102205, China.
¶¶ To whom correspondence may be addressed: Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
To whom correspondence and reprint requests may be
addressed: Institute of Neuroscience, 320 Yue-Yang Rd., Shanghai
200031, China. Tel.: 86-21-5492-1801; Fax: 86-21-5492-1801; E-mail:
zzhou@ion.ac.cn.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M210200200
2 L.-L. He and Z. Zhou, unpublished data.
3 L.-L. He and Z. Zhou, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
-BtX,
conotoxin BtX;
ChTX, charybdotoxin;
IbTx, iberiotoxin;
HPLC, high
performance liquid chromatography;
RACE, rapid amplification of the
cDNA end;
TPCK, tosylphenyl-chloromenthyl ketone;
TEA, tetraethylammonium chloride;
MS, mass spectrometry;
MALDI, matrix-assisted laser desorption ionization;
TOF, time of flight;
BK, voltage-dependent big conductance K(Ca2+)
channel;
SK, small conductance K(Ca2+) channel;
RACC, rat
adrenal chromaffin cell;
PTH, phenylthiohydantoin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hille, B. (1992) Ionic Channels in Excitable Membranes , Sinauer Associates Inc., Sunderland, MA |
2. | Miller, C. (1995) Neuron. 15, 5-10[Medline] [Order article via Infotrieve] |
3. | Zhou, Z., and Neher, E. (1993) J. Physiol. (Lond.) 469, 245-273[Abstract] |
4. | Horn, R., and Marty, A. (1988) J. Gen. Physiol. 92, 145-159[Abstract] |
5. |
Garcia, M. L.,
Knaus, H. G.,
Munujos, P.,
Slaughter, R. S.,
and Kaczorowski, G. J.
(1995)
Am. J. Physiol.
269,
C1-C10 |
6. | Hoshi, T., and Aldrich, R. W. (1988) J. Gen. Physiol. 91, 73-106[Abstract] |
7. | Myers, R. A., Cruz, L. J., Rivier, J. E., and Olivera, B. M. (1993) Chem. Rev. 93, 1923-1936 |
8. | McIntosh, J. M., Olivera, B. M., and Cruz, L. J. (1999) Methods Enzymol. 294, 605-624[Medline] [Order article via Infotrieve] |
9. | Le Gall, F., Favreau, P., Richard, G., Benoit, E., Letourneux, Y., and Molgo, J. (1999) Belg. J. Zool. 129, 17-42 |
10. | Adams, D. J., Alewood, P. F., Craik, D. J., Drinkwater, R., and Lewis, R. J. (1999) Drug Dev. Res. 46, 219-234[CrossRef] |
11. | Olivera, B. M. (1999) J. Comp. Physiol. A Sens. Neural Behav. Physiol. 185, 353-359[Medline] [Order article via Infotrieve] |
12. |
Olivera, B. M.,
Walker, C.,
Cartier, G. E.,
Hooper, D.,
Santos, A. D.,
Schoenfeld, R.,
Shetty, R.,
Watkins, M.,
Bandyopadhyay, P.,
and Hillyard, D. R.
(1999)
Ann. N. Y. Acad. Sci.
870,
223-237 |
13. | Olivera, B. M., and Cruz, L. J. (2001) Toxicon 39, 7-14[CrossRef][Medline] [Order article via Infotrieve] |
14. | Olivera, B. M., Rivier, J., Clark, C., Remilo, C. A., Corpuz, G. P., Abogadie, F. C., Mena, E. E., Woodward, S. R., Hillyard, D. R., and Cruz, L. J. (1990) Science 249, 257-263[Medline] [Order article via Infotrieve] |
15. | Chen, J. S., Fan, C. X., Hu, K. P., Wei, K. H., and Zhong, M. N. (1999) J. Nat. Toxins 8, 341-347[Medline] [Order article via Infotrieve] |
16. | Johns, M. B., Jr., and Paulus-Thomas, J. E. (1989) Anal. Biochem. 180, 276-278[Medline] [Order article via Infotrieve] |
17. |
Zhou, Z.,
and Misler, S.
(1995)
J. Biol. Chem.
270,
3498-3505 |
18. | Wu, J. J., He, L. L., Zhou, Z., and Chi, Z. W. (2002) Biochemistry 41, 2844-2849[CrossRef][Medline] [Order article via Infotrieve] |
19. | Colquhoun, D., and Hawkes, A. G. (1995) in Single Channel Recording (Sakmann, B. , and Neher, E., eds), Second Ed. , pp. 397-482, Plenum Publishing Corp., New York |
20. |
Xia, X. M.,
Ding, J. P.,
and Lingle, C. J.
(1999)
J. Neurosci.
19,
5255-64 |
21. | Lawson, K. (1996) Clin. Sci. 91, 651-663[Medline] [Order article via Infotrieve] |
22. | Fenwick, E. M., Marty, A., and Neher, E. (1982) J. Physiol. (Lond.) 331, 599-635[Medline] [Order article via Infotrieve] |
23. |
Elhamdani, A.,
Zhou, Z.,
and Artalejo, C. R.
(1998)
J. Neurosci.
18,
6230-6240 |
24. | Neely, A., and Lingle, C. J. (1992) J. Physiol. (Lond.) 453, 97-131[Abstract] |
25. | Solaro, C. R., Prakriya, M., Ding, J. P., and Lingle, C. J. (1995) J. Neurosci. 15, 6110-6123[Abstract] |
26. | Neely, A., and Lingle, C. J. (1992) J Physiol (Lond.) 453, 133-166[Abstract] |
27. | Park, Y. B. (1994) J Physiol. (Lond.) 481, 555-570[Abstract] |
28. | Latorre, R., Vergara, C., Stefani, E., and Toro, L. (2000) Pharmacology of Ionic Channel Function: Activators and Inhibitors in Handbook of Experimental Pharmacology (Endo, M. , Kurachi, Y. , and Mishina, M., eds), Vol. 147 , pp. 197-223, Springer-Verlag, New York |
29. | McManus, O. B., Harris, G. H., Giangiacomo, K. M., Feigenbaum, P., Reuben, J. P., Addy, M. E., Burka, J. F., Kaczorowski, G. J., and Garcia, M. L. (1993) Biochemistry 32, 6128-6133[Medline] [Order article via Infotrieve] |
30. | Terlau, H., Shon, K.-J., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151[CrossRef][Medline] [Order article via Infotrieve] |
31. | Park, C. S., and Miller, C. (1992) Neuron 9, 307-313[Medline] [Order article via Infotrieve] |
32. |
Wallner, M.,
Meera, P.,
and Toro, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4137-4142 |