From the Shemyakin and Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Sciences, Ul.
Miklukho-Maklaya, 16/10, 117997, GSP-7, Moscow, Russia, the
¶ Department of Pharmacology, University College London, Gower
Street, London WC1E 6BT, United Kingdom, the
Department of
Medical Physiology, The Panum Institute, University of Copenhagen,
Denmark, and the ** NeuroSearch A/S, 93 Pederstrupvej,
DK-2750 Ballerup, Denmark
Received for publication, July 7, 2000, and in revised form, November 30, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The isolation of the peptide inhibitor of M-type
K+ current, BeKm-1, from the venom of the Central
Asian scorpion Buthus eupeus has been described previously
(Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A.,
Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning,
expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the
rapid amplification of cDNA ends polymerase chain reaction
technique from mRNA obtained from scorpion telsons. Sequence
analysis of the cDNA revealed that the precursor contains a signal
peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom
potassium channel blockers and represents a new subgroup of these
toxins. The recombinant BeKm-1 was produced as a Protein A fusion
product in the periplasm of Escherichia coli. After
cleavage and high performance liquid chromatography purification,
recombinant BeKm-1 displayed the same properties as the native toxin.
Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated,
purified, and characterized. Recombinant wild-type BeKm-1 and the three
mutants partly inhibited the native M-like current in NG108-15 at 100 nM. The effect of the recombinant BeKm-1 on different
K+ channels was also studied. BeKm-1 inhibited hERG1
channels with an IC50 of 3.3 nM, but had no
effect at 100 nM on hEAG, hSK1, rSK2, hIK, hBK,
KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1.
Thus, BeKm-1 was shown to be a novel specific blocker of hERG1
potassium channels.
K+ channels comprise a large, diverse group of
integral membrane proteins, which are found in all cells.
K+ channels are involved in neuroendocrine secretion, cell
volume regulation, electrolyte balance, and regulation of levels of
excitability. K+ channels have been classified according to
their biophysical and pharmacological characteristic (2). Recent
molecular cloning of a large number of K+ channels has
resulted in a classification into structural classes including the
2-transmembrane K+ channels (inward rectifiers), the
4-transmembrane K+ channels (2P domain channels), and the
6-transmembrane K+ channels (voltage-dependent,
Ca2+-activated, or cyclic nucleotide gated) (see
e.g. Ref. 3).
Natural toxins are useful probes for evaluating the involvement of
K+ channels in cell activity, and for investigating
K+ channel structure and localization. In recent years,
peptide toxins that block various K+ channels with high
affinity have been purified from diverse animal venoms (see Refs. 4-6
for review).
The largest group of K+ channel peptide inhibitors is the
family of neurotoxic peptides found in scorpion venoms. These peptides block, in nanomolar concentrations, both voltage-gated and
Ca2+-activated K+ channels in a wide variety of
cell types, and generally contain 31-40 amino acid residues
cross-linked by three or four disulfide bridges (5, 7, 8). These toxins
have been intensively studied using biochemical, structural, and
electrophysiological methods. Both natural and mutated recombinant
scorpion short chain toxins have also been used to identify the pore
region (9, 10), determine the subunit stoichiometry of K+
channels (11), and elucidate the topology of the extracellular face of
the channel pore (12-16).
Despite rapid advances in the molecular biology of K+
channels, the subunit composition and the physiological role of several K+ channel subtypes are still unclear. That is why the
identification and characterization of ligands that interact
specifically with ion channels is critical not only for defining their
structural and functional organization, but also to elucidate the
contribution of specific ion channels to certain physiological phenomena.
A score of years ago Brown and co-workers (17) discovered the
M-current, a voltage-dependent potassium current in
sympathetic neurons that is suppressed by muscarinic acetylcholinic
receptor activity. M-channels open at resting potentials and are slowly activated by membrane depolarization. They play a key role in controlling repetitive firing in many neurons (and hence may be of
significance to normal cognitive function, dementia, and epilepsy), and
are regulated by a variety of G protein-coupled receptors.
The 4-kDa polypeptide BeKm-1 was previously isolated from the venom of
the Central Asian scorpion Buthus eupeus, and characterized as the first peptide inhibitor of the M-like current in NG108-15 mouse
neuroblastoma × rat glioma cells (1). It was shown that BeKm-1
affected neither transient and delayed rectifier K+ current
nor Na+ current. The molecular constituents underlying the
native M-current and M-like currents have remained an intriguing
puzzle. Recently, the ether-a-go-go gene
(EAG)1 K+ channel
was suggested to contribute to the mammalian M-channel underlying the
native M-current in neurons (18). Later, heteromeric KCNQ2/KCNQ3
channels were demonstrated to contribute to the native M-current (19).
During the last year, the M-like current described in NG108-15 cells
has been reported to contain two components: a "fast" and a
"slow," which have been suggested to be carried by KCNQ2/KCNQ3 and
channels encoded by the mouse ether-a-go-go-related gene
(mERG1), respectively (20, 21). The study of the action of
BeKm-1 on these different K+-channel types might throw
light upon the true toxin target in the cell.
In heart, both KCNQ1 channels and ERG channels play significant roles
in the repolarization of the action potential. Mutations in either
channel gene result in a prolongation of the QT-interval on the
electrocardiogram and give rise to long QT syndrome. Long QT syndrome
is a disorder that may cause syncope and sudden death resulting from
episodic ventricular arrhytmias and ventricular fibrillation. Inherited
long QT type 2 results from mutaions in the human ERG, originally
referred to as hERG. As novel members of the ERG family emerge, the
terminology hERG1 seems more appropriate.
This paper presents the total amino acid sequence of BeKm-1 deduced
from cDNA sequencing and the biological properties of the
recombinant BeKm-1 and of the three mutants containing point changes in
the COOH-terminal region of the toxin. In the present study, we have
investigated the action of recombinant BeKm-1 on native M-like current
in NG108-15 cells, and directly on cloned human KCNQ- and hERG1
potassium channels.
Purification of BeKm-1 from B. eupeus Venom
Native BeKm-1 toxin was purified from the scorpion venom as
described previously (1).
Determination of Amino Acid Sequence of BeKm-1
Disulfide bonds of the purified toxin were reduced with
dithiothreitol, and SH-groups were modified with 4-vinylpyridine (22). The modified toxin was digested with Staphylococus aureus
protease V8 (Roche Molecular Biochemicals) at 37 °C for 12-14 h in
50 mM ammonium acetate buffer, pH 4.0, at a ratio of 1 µg
of enzyme per 25 µg of toxin or with trypsin at 37 °C for 16 h in 50 mM Tris buffer, pH 8.5 (ratio enzyme:toxin 1:20).
The digests were fractionated by reverse-phase HPLC on an Ultrasphere
ODS column (2 × 250 mm) using an acetonitrile gradient in 0.1%
trifluoroacetic acid. The NH2-terminal amino acid sequence
of BeKm-1 and its internal peptides were determined by automated Edman
degradation using an Applied Biosystems Sequencer (470A protein
sequencer) on-line with the phenylthiohydantoin analyzer (120A analyzer).
Preparation of mRNA from Venom Glands
Total RNA was isolated by a guanidinium thiocyanate/phenol
chloroform method (23) from scorpion venom glands frozen in liquid nitrogen immediately after sacrificing. Poly(A)-rich RNA was prepared from total RNA by two-cycle chromatography on oligo(dT)-cellulose as
described (24).
PCR Amplification of BeKm-1 DNA, Cloning, and DNA Sequencing
RT-PCR--
Reverse transcriptase (RT) reaction was performed
with 2 µg of mRNA (pre-heated at 70 °C for 5 min) in a total
reaction volume of 20 µl of 1 × RT buffer (U.S. Biochemical).
The downstream primer used was the primer RLdT with the sequence
5'-GAGAATTCGGATCCCTGCAGAAGCTTTTTTTTTTTTTTTTT-3'. Other components of
the RT reaction were 0.5 mM of each dNTP, 1 unit/µml RNAsin, 100 units of Moloney murine leukemia virus-reverse transcriptase. The mixture was incubated for 1 h at 37 °C. 1 µl of this reaction mixture was used in 50 µl of polymerase chain reaction. PCR was carried out with denaturation for 30 s at
94 °C, annealing for 1 min at 50 °C, and extension for 1 min at
72 °C. Oligonucleotide primers used were sense T1 (codes for the predicted amino acid sequence from Arg1 to Lys6
of BeKm-1 with codon degeneracy),
5'-GGAATTCG(G/A/T/C)CC(G/A/T/C)AC(G/A/T/C)GA(C/T)ATAAA(A/G)TG-3', and
antisense RL, 5'-GAGAATTCGGATCCCTGCAGAAGCTT-3'. The 220-bp fragment was
gel purified, digested with EcoRI/PstI, and
ligated to similarly prepared vector pBluescript SK+ (Stratagene).
Escherichia coli MH1 was used for plasmid propagation. The
recombinant clones were analyzed with the standard technique (25) and
sequenced on both strands using the Sanger (26) dideoxynucleotide chain termination method.
5'-RACE-PCR--
5'-RACE was performed according to the anchored
PCR technique described (27). A poly(dG) tail sequence was introduced
to the first strand cDNA with terminal deoxynucleotidyl transferase (U. S. Biochemical) in 1 × terminal deoxynucleotidyl
transferase buffer with 1 mM dGTP for 15 min at 37 °C;
the reaction was stopped by heating at 70 °C for 10 min.
Amplification was performed using one specific primer T2 corresponding
to the 3'-flanking region of the cDNA of BeKm-1
(5'-GCATTACATACTTTCATTATAAATCTG-3') and poly(dC) primer C13
(5'-GTGAATTCCTTAACCCCCCCCCCCCC-3'). The denaturation step was at
94 °C for 30 s, the annealing step was at 57 °C for 1 min,
and the extension was at 72 °C for 1 min. Amplification was
performed for 30 cycles and the product was analyzed. An appropriate size region was cut from the gel and directly reamplified with a
mixture of universal primer M13 and M13-C13
(5'-GTAAAACGACGGCCAGTGAATTCCTTAACCCCCCCCCCCCC-3') as the primers for
the poly(dG) end at the ratio of 10:1 and the T2 primer. Amplification
was performed in the same conditions for 12 cycles. Two PCR bands
~250 and 300 bp were reamplified separately with M13 and the T3
primer, corresponding to C terminus of BeKm-1
(5'-CCATTCACGCACCTTCCATTAGTC-3'). The PCR products were cloned using
AdvanTAgeTM PCR Cloning Kit (CLONTECH)
according to the manufacturer's procedure and sequenced.
Site-directed Mutagenesis and Expression Vectors Construction
The following nucleotide probes were used for mutagenesis: R27K,
5'-CAAAAACCATT-CACGCACTTTCCATTAGTCTTCCC-3'; F32K,
5'-CGAATTCTAAAAACAGTCGCACTTACCATTCACGCAC-3'. Mutated codons are underlined. Mutagenesis of Arg27 was
achieved using two sequential polymerase chain reactions as described
(28). The cDNA encoding BeKm-1 was amplified by PCR. The forward
primer was nondegenerate oligonucleotide E1, 5'-GGAATTCGGACGACGACGACAAGCGACCTACAGATATAAAATGCAG-3',
containing an EcoRI restriction enzyme site (italicized) and
corresponding to five codons encoding an enterokinase cleavage site and
NH2-terminal residues 1-6 of BeKm-1. Primer E2,
5'-CGAATTCTAAAAACAGTCGCAAAAACCATTCACGC-3', or F32K primer in
the case of F32K-contained mutants, were used as the reverse primers.
Both of them carried an EcoRI restriction enzyme site
(italicized) and corresponded to the stop codon and COOH-terminal
residues 28-36 of BeKm-1. The PCR fragments encoding mature and
mutated BeKm-1 were gel purified, digested with EcoRI, and
cloned into the expression vector pEZZ18 (Protein A gene Fusion Vector,
Amersham Pharmacia Biotech). Clones were screened for the presence and
orientation of the inserts by PCR. The resulting constructs were
checked by sequencing and used to transform E. coli HB101
stain for protein production (29).
Expression and Purification of Recombinant Toxins
The wild type and mutated genes of BeKm-1 were expressed in the
periplasm of E. coli as a fusion protein with two
IgG-binding domains (ZZ) of staphylococcal Protein A. E. coli HB101 cells harboring the expression vectors were cultured at
37 °C in LB medium containing 100 µg/ml ampicillin. After 30 h the cells were harvested, resuspended in TS solution (50 mM Tris buffer, pH 7.6, 150 mM NaCl), and lysed
by ultrasonication. After ultrasonication, the mixture was centrifuged
for 15 min at 15,000 rpm to remove any remaining insoluble particles.
The supernatant was applied to an IgG-Sepharose 6FF column (Amersham
Pharmacia Biotech). The column was washed first with TS solution,
containing 1 M NaCl and 0.05% Tween 20 and then with TS
solution. The bound proteins were eluted with 0.5 M acetic
acid, pH 3.4, and immediately lyophilized. Purity of the hybrid
proteins was checked by SDS-polyacrylamide gel electrophoresis
(30).
The toxins were cleaved from fusion proteins by enterokinase (31) (1 µg/50 µg of fusion protein) at 37 °C in 50 mM
Tris buffer, pH 8.0, for up to 36 h. The recombinant toxins were
purified from the cleavage mixture by chromatography on a reverse phase HPLC column (Delta Pak C18 300-Å pore, 3.9 × 300 mm,
Waters) using an acetonitrile gradient in 0.1% trifluoroacetic acid.
The fractions containing recombinant toxins were rechromatographed on
an ODS Ultrasphere column (4.6 × 150 mm, Beckman). Mass
spectrometry and NH2-terminal amino acid determination
verified the composition of the purified material. The peptide content
was determined using the bicincholinic acid method (32) with bovine
serum albumin as the standard.
Mass Spectrometry
Mass analysis of the recombinant toxins was performed in a
VISION 2000-time of flight mass spectrometer with matrix-assisted laser
desorption ionization, Thermo Bioanalysis Corp. (United Kingdom).
Electrophysiology
Measurement of M-type Currents in NG108-15 Cells--
NG108-15
mouse neuroblastoma × rat glioma cells were cultured and
differentiated as described previously (32). Recordings were made in
the whole cell configuration of the patch clamp, at room temperature
(20-25 °C). The perfusing solution comprised (mM): NaCl
144, KCl 2.5, CaCl2 2, MgCl2 0.5, HEPES 5, and
glucose 10, adjusted to pH 7.4 with Tris base Electrodes (2-4 M
To record the M-like current, cells were voltage-clamped with
discontinuous ("switching") amplifier (Axoclamp-2A, Axon
Instruments, Inc.) with sampling a voltage at 6-8 kHz (50% duty
cycle). Commands were generated via Digidata 1200 interface using
pClamp 6 software (Axon Instruments). A standard voltage step protocol
for M-current recording was used (33-35), in which cells were held at
The resulting numerical data for control and BeKm-1-inhibited current
records were processed in Quattro Pro version 5.0, and percentage
reduction of the current at Stably and Transiently Transfected HEK-293 Cell
Lines--
HEK-293 cells were incubated for 3-5 h in Opti-MEM medium
with a transfection mixture containing: 2.0 µg of the appropriate K+ channel cDNA ( Measurements in HEK-293 Cells--
The patch-clamp set-up and
whole cell recordings were as previously described (36, 37). SK, IK,
and BK currents were recorded after application of voltage ramps
ranging from
Cells expressing KCNQ2/KCNQ3 channels were bathed in an extracellular
Na+ solution and the currents were activated by a 1-s step
from a holding potential of Solutions Used in HEK-293 Cells--
The composition of
solutions used in experiments performed on HEK-293 cells consisted of
extracellular Na+ solution (mM): NaCl 140, KCl
4, CaCl2 2, MgCl2 1, and Hepes 10 (pH 7.4, titrated with NaOH), the extracellular K+ solution
(mM): KCl 144, CaCl2 2, MgCl2 1, and Hepes 10 (pH 7.4, titrated with KOH); and the intracellular
solutions (mM): KCl 110, CaCl2 5.1-7.6,
MgCl2 1.2-1.4, Na2ATP 4, EGTA 10/KOH 30, and Hepes 10 (pH 7.2, titrated with KOH).
Amino Acid Sequence of BeKm-1--
The partial amino acid sequence
of BeKm-1 was determined from the NH2-terminal sequence and
the sequences of the peptides derived from proteolytic digestion. The
sequence of the NH2-terminal amino acid fragment was
obtained after reducing and modification of cysteine residues. The
hydrolysis of the modified toxin chain using V8 proteinase from
S. aureus resulted in two peptides, which were sequenced.
The sequences of these peptides had an overlapping region, and the
primary structure of the BeKm-1 fragment
Arg1-Cys28 was determined. Amino acid
sequences of some peptides obtained from tryptic hydrolysis of native
BeKm-1 corresponded to the established regions of the toxin molecule.
Cloning and Sequencing of BeKm-1 cDNA--
To determine the
full amino acid sequence and to predict the structure of the toxin
precursor, the cDNA encoding BeKm-1 was isolated. Briefly, the
first strand cDNA was synthesized using poly(A)-rich RNA by Moloney
murine leukemia virus reverse transcriptase and first-strand primer
(RLdT) containing restriction sites for cloning. Degenerate
oligonucleotide primer T1 was designed using amino acid sequence
information and codes for the toxin region from Arg1 to
Lys6.
PCR using RL and T1 primers yielded a single band of the expected size
(220 bp). This PCR product was subcloned in pBluescript between the
PstI and EcoRI sites and sequenced. All cDNA
sequences had one major open reading frame (ORF), encoding an amino
acid sequence corresponding to the partial amino acid sequence of
BeKm-1 obtained from primary structural analysis of the native toxin.
5'-RACE was used to identify the unknown sequence of the 5'-end of the
mRNA. 5'-Flanking sequence information was obtained using the
anchored PCR technique (27). In this procedure, mRNA was first
transcribed with reverse transcriptase and a poly(dG) tail was added to
the 3'-end of the strand with terminal deoxynucleotidyl transferase.
The product was then amplified with a specific 3'-primer (in our case a
T2 oligonucleotide) and another oligonucleotide consisting of a
poly(dC) tail (C13 and M13-C13 primers). Series of consistent PCR
generated two bands of about 300 and 250 bp with cloned cDNA
sequences that had long and short 5'-untranslated region, respectively,
that may be a result of partial degradation of the mRNA. The
full-length cDNA of BeKm-1, together with its 5'- and
3'-untranslated region, is shown in Fig.
1.
Analysis of the cDNA Sequence--
The full-length of the
cDNA, excluding the poly(A) tail, was 365 bp and contained a 171-bp
ORF encoding a 57-amino acid peptide. The first ATG was located at
position 121. The 3'-untranslated region of the cDNA contained a
putative polyadenylation signal (AATAAA) ~16 nucleotides upstream
from the poly(A) tail. The ORF encoded a polypeptide precursor for
BeKm-1 in which the first 21 amino acid residues are predicted to be a
signal peptide followed by the mature 36-amino acid peptide. The signal
peptide has structural features characteristic of secreted proteins,
and shows homology to the published leader peptides of KTx, KTx2 (39),
and Ts Expression of Recombinant BeKm-1 and Its Mutants--
To obtain
significant quantities of the peptide and its mutated forms for
structure-function investigation, the E. coli expression system was adopted. We expressed the wild-type BeKm-1 and three mutated
toxins (R27K, F32K, and R27K/F32K) as fusion proteins with two
IgG-binding domains of Protein A from S. aureus. Sense and
antisense primers, both with a specific restriction enzyme site, were
used to amplify the BeKm-1 toxin cDNA for cloning into the pEZZ18
expression vector. The pentapeptide sequence DDDDK, which is recognized
by the restriction protease enterokinase, was inserted immediately
upstream of the BeKm-1 sequence. A translation termination codon was
inserted at the end of the BeKm-1 cDNA. Site-directed mutants were
constructed using PCR, and each mutated plasmid was verified by
sequencing. The final constructs were transformed into the E. coli strain HB101. Fusion proteins were directly secreted into the
periplasm of HB101, making them easy to purify by affinity
chromatography on an IgG-Sepharose column. The size of the affinity
purified proteins observed from SDS-polyacrylamide gel electrophoresis
was in accordance with that expected from a fusion protein of ZZ and
BeKm-1. The yield of fused toxins varied from 2 to 6 mg/liter of
culture. The fusion proteins were treated with enterokinase and
purified by reverse-phase HPLC to obtain the pure recombinant toxins.
The recombinant, enterokinase-digested BeKm-1 had the same retention
time as the native toxin when fractionated by reverse-phase HPLC (Fig.
2). Homogeneity of recombinant BeKm-1 and
of each mutant was further confirmed by analytical reverse-phase HPLC
employing an Ultrasphere ODS column. The total yield of purified recombinant peptides was ~200 µg of toxin/liter of culture. The molecular mass of the recombinant products obtained by matrix-assisted laser desorption ionization mass spectrometry were in accordance with
the expected theoretical mass calculated from sequence data (determined
mass 4092, 4064, 4074, and 4046; calculated mass 4091.69, 4063.67, 4072.69, and 4044.68 Da for the recombinant wild-type and native
toxins, mutants R27K, F32K, and R27K/F32K, respectively).
Effect of the Recombinant BeKm-1 and of the Mutants on the M-like
Current in NG108-15 Cells--
The effect of the recombinant BeKm-1
and all mutated toxins were tested for inhibition of the M-like current
in mouse neuroblastoma × rat glioma NG108-15 cells. The M-like
current in NG108-15 cells is a sustained, voltage-activated, outward
potassium current observed at potentials positive to
The recombinant BeKm-1 toxin clearly inhibited the M-like current by
reducing the standing (holding) outward current at The Effect of BeKm-1 on Cloned Human KCNQ and hERG1
Channels--
To elucidate the molecular composition of the current
blocked by BeKm-1, the toxin was applied to HEK-293 cells transiently expressing human KCNQ2/KCNQ3 channels, which have been suggested to
underlie the M-current (19). In whole cell experiments, the KCNQ2/KCNQ3
current was visualized as an outward current activating slowly upon a
voltage step from
Since the M-like current in NG108-15 cells has been reported to include
both a KCNQ and an ERG component (20, 21), BeKm-1 was also tested on
human ERG1 channels. In preliminary experiments, BeKm-1 was added at a
concentration of 100 nM to a HEK-293 cell line stably
transfected with hERG1 and KCNE1. BeKm-1 inhibited the hERG1 + KCNE1
current activated by a cardiac action potential protocol (see
"Experimental Procedures"). The block was fast and reversible with
an IC50 value estimated at 10-30 nM
(n = 7, data not shown). The three mutants of BeKm-1
inhibited the hERG1 + KCNE1 current with similar potencies
(n = 3 for each toxin-mutant, data not shown).
The inhibition of hERG1 by BeKm-1 was characterized further in HEK-293
cells transiently transfected with the hERG1-cDNA alone. Fig.
4A shows a whole cell
experiment with 144 mM [K+]0,
where the hERG1 current was fully activated by stepping to +40 mV for
400 ms every 5 s from a holding potential of
Two Specificity of the BeKm-1 Toxin--
The specificity of
BeKm-1 was tested at a variety of cloned K+ channels and,
as stated in Table I, addition of 100 nM of the toxin in whole cell experiments was without
effect at four members of the family of calcium-activated potassium
channels. These channels were activated by 100-300 nM free
calcium in the pipette solution and a ramp-protocol ranging from
To determine whether the BeKm-1 toxin is a general blocker of the
ether-a-go-go family, or if it is specific to hERG1, the toxin was tested on the cloned hEAG and rELK1 channels. Whole cell
experiments on transiently transfected HEK-293 cells were performed in
140 mM [K+]o solution applying the
same protocol as in the experiments of the hERG1 channel in Fig.
4A. To prevent blockade of the hEAG channel by intracellular
Ca2+ (42), no CaCl2 was added to the pipette
solution in these experiments. When performing recordings on rELK1
expressing HEK cells, the holding potential was set to The discovery of high-affinity K+ channel toxins has
increased the understanding of the K+ channel structure,
distribution, and subcellular localization in different cell types
significantly. In this study we investigated the structure and function
of BeKm-1, a toxin from venom of the scorpion B. eupeus (1).
It is demonstrated here that BeKm-1 is a novel specific blocker of the
hERG1 potassium channel (IC50 = 3.3 nM) with
potency 5 times higher than that of the previously reported peptide
ERG-Tx (44). Recombinant BeKm-1, like the native toxin (1), also
reduced the M-like current in NG108-15 mouse neuroblastoma × rat
glioma cells, part of which is carried by mERG1 channels (20, 21). It
did not, however, inhibit KCNQ channels, in agreement with the lack of
effect of the native toxin on the KCNQ2/KCNQ3 M-currents in rat
sympathetic neurons (1).
Amino Acid Alignment Analysis--
The combination of protein
chemistry methods and the RT-PCR technique enabled us to obtain the
primary structure of BeKm-1. BeKm-1 is a 36-amino acid peptide
containing 6 cysteines and 6 positively charged residues.
On the basis of analysis of its amino acid sequence, BeKm-1 belongs to
a family of closely related scorpion toxins. The amino acid sequence of
BeKm-1 was compared with those of other known scorpion peptide blockers
of K+ channels (Fig. 5). The
largest degree of amino acid identity is observed between BeKm-1, IbTx,
and TsTx K
The functional role of the highly conserved Lys27 was
previously studied by individual chemical modification and by
site-directed mutagenesis of ChTx. It has been identified that the
positively charged Lys27 is an important toxin element
implicated in the recognition of K+ channels, and the
critical residue for voltage-dependent block of the BK
channel (50) and Shaker K+ channel (51).
Furthermore, even a minor change of a functionally important
Lys27, such as mutation to Arg, was unfavorable. For
example, in the Shaker channel, a conservative mutation of
this lysine to arginine destabilized ChTx binding over 1000-fold (39).
Arginine instead of lysine in a position equivalent to 27 was found
only in Pi7 toxin from scorpion Pandinus imperator. This
toxin, however, was absolutely inactive on the Shaker B
channel, whereas the highly homologous toxin Pi4 from the same venom
containing a lysine residue completely blocked this type of
K+ channel (53).
On the other hand, BeKm-1 displays no homology with ergtoxin (ERG-Tx)
isolated from the scorpion Centruroides noxius (44). ERG-Tx
has an another structure (Fig. 5) that differs from those previously
known for K+ channel toxin blockers. ERG-Tx specifically
inhibits only ERG channels with IC50 of 16 nM.
It is interesting that ERG-Tx and BeKm-1 have only two homologous
residues, namely Arg and Asp (positions 1 and 4 in BeKm-1 and 2, 3 in
ERG-Tx) in the NH2-terminal part of the toxins. As
Arg1 in BeKm-1 is unique among all K+ channel
blockers from scorpion venom, it may have an important role in hERG1
channel inhibition.
Choice of the Mutations--
Previously, it was suggested that the
highly conserved cluster of about 10 amino acid residues in the
COOH-terminal region of scorpion toxins may play a prominent role in
toxin-channel interactions. We hypothesized that the COOH-terminal
26-32 sequence of BeKm-1 may contribute the core of the toxin-binding
site that recognizes K+ channels. To elucidate this point,
site-directed mutagenesis of BeKm-1 was undertaken. The DNA segment
encoding BeKm-1 was mutated to obtain a set of variants of the toxin to
study structure-function relationships. Mutations were chosen so as to
produce a change in the original biological function. Two amino acids,
Arg27 and Phe32, were substituted by the Lys
found in most K+ channel toxins. Since Lys27 is
highly conserved among all the toxins and has an important role in
K+ channel inhibition, it was interesting to assess its
contribution to the block of hERG1 channels by BeKm-1.
Phe32 has not been found in other potassium channel
blockers from scorpion venom, suggesting that its role may be important
for hERG1 inhibition. The double mutant R27K/F32K had the same sequence
of active region as many others scorpion toxins.
Expression of Recombinant Toxins--
We successfully produced
BeKm-1 and its altered analogues in the periplasm of E. coli, using the plasmid pEZZ18 as a fusion protein with two
IgG-binding domains of Protein A from S. aureus. Previous
studies showed that this type of expression system is particularly well
adapted to disulfide-rich proteins. It has been used to express
erabutoxin A (54) and BotXIV, insect-specific
Heterologous expression of cDNAs encoding other small scorpion
toxins, like charybdotoxin (ChTx) from Leiurus
quinquestriatus (56) and margatoxin (MgTx) from Centruroides
margaritatus (57) has been described in E. coli. The
fusion toxins with the gene 9 protein of T7 bacteriophage produced in
the cytoplasm were cleaved by factor Xa after purification and the
resulting toxins refolded in vitro. The major drawback of
the production procedure is that the recovery yield is rather low as
compared with those reached using the procedure mentioned above.
Recently KTx2 was successfully produced as a fusion protein with
maltose-binding protein in the periplasm of E. coli (58)
with a yield comparable to that obtained in the present study.
Effect on K+ Channels--
Experiments presented here
demonstrate that recombinant BeKm-1 has structural and functional
characteristics identical to those of the native toxin. Recombinant
BeKm-1 inhibits the M-like K+ current in NG108-15 cells in
a manner very similar to that of the native toxin (see Ref. 1). These
results indicate that the recombinant and native toxins are
functionally identical in their binding properties. Furthermore, all
mutants inhibited the M-like potassium current in NG108-15 cells
similar to that of the recombinant wild-type BeKm-1, although the
mutant R27K was found to be marginally less potent than BeKm-1.
The examination of the action of BeKm-1 on the cloned K+
channels revealed that BeKm-1 potently blocked the human ERG1 channel. Coexpression of the hERG1 channel with its putative
Point substitutions in the COOH-terminal region of BeKm-1 did not
change the efficacy of the toxin to block M-like currents or cloned
hERG1 channels indicating that the BeKm-1 mutants have the same
affinity for hERG1 channels as the wild-type toxin. Therefore, the
highly conserved sequence found in K+ channel-selective
scorpion toxins, which constitutes a common high affinity binding site
recognizing both voltage-gated and Ca2+-activated
K+ channels, is not essential for ERG channels. The high
affinity of the BeKm-1 toxin and the specificity toward hERG1 channels might result from interactions different from other pore-blocking toxins and probably involve the NH2-terminal toxin region.
Further efforts will include this subject of analysis.
By using expression systems in combination with site-directed
alterations in the peptide sequence, the molecular basis of BeKm-1
recognition and inhibition of ERG channels may be addressed. With the
feasibility of producing large amounts of BeKm-1 in vitro, it will be possible to study the three-dimensional structure of the
peptide. The study of the interaction of BeKm-1 with its channel receptor promises to reveal new information regarding ERG channels structure and function, and to clarify how scorpion toxins act on
diverse members of the K+ channel superfamily. Finally,
BeKm-1 provides a specific tool not only to determine the functional
role that ERG channels play in target tissues or to develop the
molecular pharmacology of this channel, but also to determine the
subcellular localization of this channel in heart and brain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
were filled with a solution containing (mM): K acetate 90, KCl 20, Hepes 40, MgCl2 3, EGTA 3, Na2ATP 3, and Na-GTP 0.3, adjusted to pH 7.2 with NaOH. Recombinant and mutated
BeKm-1 were dissolved in water at 100 µM, and added to
the circulating bath solution to give a final concentration of 100 nM.
28 mV and stepped for 1 s to potentials between
18 and
128
mV, by 10-mV negative increments. Current amplitudes at the end of each step were measured for subsequent construction of current-voltage relations using pClamp 6 software. The leak component of current was
estimated by extrapolating a line fitted by eye from the region of the
current-voltage relationship (negative to
60 mV) where only linear
(ohmic) currents were observed.
28 mV was calculated. Graphs were created
in Microcal Origin version 4.1. In addition, the current was monitored
during toxin application by repeated steps from
28 mV to
68 and
108 mV, to ascertain when a steady-state block was reached.
-subunits: KCNQ2-KCNQ4, hEAG,
rELK1, and hERG1;
-subunits: KCNE1 and KCNE2), LipofectAMINE (Life
Technologies), and Plus reagent (Life Technologies). In experiments
with transiently transfected cells, recordings were performed 24-72 h
post-transfection using enhanced green fluorescent protein as a
marker of successful transfection. Some experiments were performed on
HEK-293 cells stably expressing K+ channels (hSK1, rSK2,
hIK, hBK, or hERG1+KCNE1). For details see Refs. 36-38.
80 mV to +80 mV (duration 200 ms, holding potential 0 mV). The bath solution was an extracellular K+ solution.
90 mV to
30 mV. In the initial
experiments with the hERG1 expressing cells, the currents were
activated by a voltage protocol reassembling a cardiac action
potential. In the protocol the cells were held at
90 mV and
depolarized to +30 mV followed by 8 hyperpolarizing ramps altogether
shaping an action potential with a duration of 315 ms.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (79K):
[in a new window]
Fig. 1.
Nucleotide sequence of the cDNA encoding
the BeKm-1 precursor. ORF sequence is boxed; open
boxes represent the leader sequence, followed by filled-in
box carrying the nucleotide sequence of the mature BeKm-1 toxin.
The deduced amino acid sequence of the ORF is indicated by the
three-letter code below the appropriate nucleotide codon.
The putative polyadenylation signal AATAAA is
underlined.
(40), K+ channel blockers from scorpion venoms.
View larger version (18K):
[in a new window]
Fig. 2.
Reverse phase HPLC of recombinant and native
BeKm-1. A, sample of fusion protein digested with
enterokinase (1 µg of enterokinase/50 µg of fusion protein in 50 mM Tris buffer, pH 8.0, for 30 h at 37 °C);
B, native BeKm-1. Column C18 Delta Pak 3.9 × 300 mm, 300-Å pore (Waters). Elution was performed with a gradient
of solvent B (0.1% trifluoroacetic acid in acetonitrile) in solvent A
(0.1% trifluoroacetic acid in water) at flow rate 2 ml/min.
70 mV (33). This
current is similar to the M-current of sympathetic ganglia in many
respects, but differs from it in certain pharmacological properties
(33), so we refer to it here as an "M-like" current. We used the
standard protocol for M-like current recording (for example, see Refs. 33 and 35). The cell was voltage-clamped at holding potential of
28
mV, to pre-activate the M-like current, and the current was then
deactivated by 1-s voltage steps to more hyperpolarized levels (see
also "Experimental Procedures").
28 mV holding
potential, the amplitude of its relaxation on deactivating and the
absolute current amplitude at the end of the steps (Fig. 3A). Fig. 3B shows
the inhibition of the current at
28 mV by 100 nM
recombinant BeKm-1 (n = 3) and mutants R27K, F32K, and R27K/F32K (n = 4 for each). A one-tailed t
test (Quattro Pro) gave a marginal significance (p = 0.049) in favor of the hypothesis that mutant R27K had marginally lower
activity that the wild-type toxin. Nevertheless, its blocking activity
was still appreciable at >40%. Inhibition by the other two mutants
did not differ significantly from that by the nonmutated toxin; all
were >50%. The results demonstrate that the blocking effect of the
recombinant peptides on the M-like current in NG108-15 cells is not
less than that of the native toxin (about 45% (see Ref. 1)). Since the
M-like current in NG108-15 cells has been suggested to be carried by both KCNQ2/KCNQ3 and mErg1 channels (20, 21), the partial blocking
effect of the toxin could mean that the toxin blocked selectively only
one of the component channels.
View larger version (28K):
[in a new window]
Fig. 3.
Effect of the recombinant BeKm-1 and mutants
on the M-like current in neuroblastoma × glioma NG108-15
cells. A, inset records show an example of current
responses of a cell to a series of voltage steps from a holding
potential of 28 mV to command potentials between
18 and
128 mV in
10 mV increments, recorded before and 3 min after starting superfusion
with 100 nM recombinant BeKm-1. Dotted line
indicates zero current. An arrow indicates were current was
measured for a graph. The graph shows the absolute current levels
(current measured from zero current) attained at the end of each
voltage step, plotted against the command potential, before and during
superfusion with toxin. B, the columns show the mean
percentage inhibition of M-like currents by 100 nM
recombinant BeKm-1 and the three mutated variants. The bars
show the standard error of the mean in each case, n is
number of experiments. Of the differences between mean values, only
that between recombinant BeKm-1 and mutant R27K attained marginal
statistical significance. M-like current amplitudes were measured at
28 mV after leak subtraction from the current-voltage curves obtained
as in A (see also "Experimental Procedures").
90 to
30 mV. The step was repeated every 5 s
and after establishment of a stable baseline, the Na+
solution was changed to a Na+ solution containing 100 nM BeKm-1. The presence of BeKm-1 in the bath solution for
2-3 min did not change the amplitude of the current (n = 3, data not shown). The toxin was tested in a similar way on other
members of the KCNQ family, KCNQ4 (n = 3, not shown)
and KCNQ1 coexpressed with KCNE1 (n = 3, not shown). BeKm-1 did not modulate any of these currents.
80 mV. After each
step the voltage was returned to
120 mV and large inward tail
currents could be recorded. Characteristic of hERG1, relief from
channel inactivation is apparent at the peak of the tail currents
shown. In Fig. 4B the size of the tail currents is plotted
as a function of time (same experiment as in Fig. 4A). The
hERG1 current was blocked as 4 nM and subsequently 100 nM BeKm-1 was added to the bath solution for the periods
indicated by bars. After wash out of the toxin for 2 min, the tail
current recovered totally and reached the control level. Fig.
4C depicts the mean of normalized tail currents as a
function of the BeKm-1 concentration from four experiments. The
IC50 values for the effect of BeKm-1 on the hERG1 channel
was calculated by fitting the normalized tail currents to the function
Itail = 1
([BeKm-1]n/[BeKm-1]n + IC50), where
Itail is the fraction of unblocked current and n is the Hill coefficient. From the fitted dose-response
curve an IC50 value of 3.3 nM and a Hill
coefficient of 0.9 was obtained (n = 4, error
bars represents S.E.).
View larger version (14K):
[in a new window]
Fig. 4.
The effect of BeKm-1 on the cloned hERG1
channel. A, whole cell current traces recorded from HEK
cells transfected with the cDNA encoding hERG1. The experiment was
performed with high K+ solutions on both sides of the
membrane at a holding potential of 80 mV. Tail currents were elicited
by applying a +40 mV pre-pulse followed by a step to
120 mV for 500 ms applied every 5 s. The traces represent recordings at
superfusion with high K+ solution alone, in the presence of
4 nM, and 100 nM BeKm-1, respectively.
B, time course of the same experiment as in A.
Current was measured at the arrow shown in A. C,
mean dose-response curve from four experiments (error bars
represent S.E.). The points were fitted with a modified Hill
equation.
-subunits, KCNE1 and KCNE2, are known to interact with the hERG1
channel (41). To test if association of the hERG1
-subunit with its
-subunits would influence the effect of BeKm-1, co-transfection
studies were performed. 10 nM BeKm-1 was applied to HEK
cells transiently transfected with hERG1 + KCNE1 and hERG1 + KCNE2,
respectively. All experimental conditions was as in Fig. 4A.
Kd values were estimated by exponential fitting to the time course of blockade (37). The Kd values 5 and 7 nM obtained from recordings with hERG1 + KCNE1 and
hERG1 + KCNE2 were not found to be significantly different from the
block by BeKm-1 on hERG1 channel alone.
80
to +80 mV was used. The hSK1, rSK2, and hIK channels are not
voltage-dependent, and weakly inward rectifying currents
are measured upon application of voltage ramps with a 140 mM K+ solution in the bath (see Refs. 37 and
38). The voltage-dependent hBK channels were activated at
potentials more positive than +30 mV. After the addition of 100 nM BeKm-1 and a washing period, the specificity of the
current measured in each of the experiments was verified by the
inhibition induced by apamin (hSK1 and rSK2), charybdotoxin (hIK), or
iberiotoxin (hBK) at 100 nM.
Specificity of BeKm-1
80 to +80 mV, 200 ms
duration). The KCNQ1/KCNE1, KCNQ2/KCNQ3, or the KCNQ4 current was
visualized as an outward current activating slowly upon a voltage step
from
90 to
30 mV recorded in asymmetrical Ringer's solution (high
extracellular Na+, high intracellular K+). The step was
repeated every 5 s and after establishment of a stable baseline,
100 nM was applied, and the current amplitude recorded. The
ether-a-go-go like channel experiments were performed with
high K+ solutions on both sides of the membrane at a holding
potential of
80 mV. Tail currents were elicited by applying a +40 mV
pre-pulse followed by a step to
120 mV for 500 ms applied.
100 mV, due to
the low threshold activation of these channels (43). The inward tail
currents were followed on-line while the cells were superfused with 100 nM BeKm-1 for 3-6 min. No effect was observed on the hEAG
tail current (n = 3), however, the rELK1 current was
slightly inhibited by the toxin. The tail current recorded in the
presence of 100 nM toxin was reduced by 9.4 ± 2.6%
(n = 3, S.D) as compared with the control current level.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(40.5%). The conserved position of the 6 cysteine
residues among all toxins suggests a common disulfide bridging pattern
as shown for ChTx, IbTx, MgTx, KTx, and other scorpion toxins, whose
spatial structures were resolved by NMR (45-48). It is possible that
the folding and general conformation of BeKm-1 is homologous to the
previously determined folding of scorpion venom toxins. Despite the
homology between BeKm-1 and other scorpion toxins, it cannot be
assigned to one of the 12 distinct subfamilies of scorpion
K+ channel blocking peptides proposed by Tytgat (49). Thus,
BeKm-1 belongs to a new subfamily of scorpion toxins. Comparison of the deduced amino acid sequence of BeKm-1 with those of other
K+ channel-specific scorpion toxins revealed that BeKm-1
displays several important sequence differences, which could explain
its specific effect on the hERG1 K+ channel. The principal
differences are in the COOH-terminal part that is highly conserved
among all scorpion toxins. BeKm-1 contains Arg27,
Val29, and Phe32 instead of Lys, Met, and Lys,
respectively.
View larger version (33K):
[in a new window]
Fig. 5.
Sequence homology between BeKm-1 and other
scorpion toxins acting on K+ channel.
Representatives of some -KTx subgroups (49): ChTx, charybdotoxin
from Leiurus quinquestriatus hebraeus; IbTx, iberiotoxin
from Buthus tamulus; TsTx-K
, tityustoxin K
from
Tityus serrulatus, NxTx, noxiustoxin from C. noxius; KTx, kaliotoxin from Androctonus mauretanicus
mauretanicus; ScTx, scyllotoxin from Leiurus quinquestriatus
hebraeus, Pi1 pandinotoxin 1 from P. imperator.
Sequences are aligned at Cys residues (boxes). Identical
residues are filled. The last line is the ERG
channels inhibitor ERG-Tx, ergtoxin from C. noxius
(44).
toxin of the scorpion
Buthus occitanus tunetanus (55). The combination of
secretion and single step affinity purification has obvious advantages.
The yield of the desired fusion product was 2-6 mg/liter of culture,
giving about 200 µg of recombinant toxin after cleavage from the
fusion protein by enterokinase and purification by HPLC.
-subunits, KCNE1
and KCNE2, did not change the toxin affinity. The results show that
recombinant BeKm-1 does not interact with hEAG, hSK1, rSK2, hIK, hBK,
KCNQ1/KCNE1, KCNQ2/KCNQ3, or KCNQ4 channels. As BeKm-1 did not affect
KCNQ2/KCNQ3 channels it is clear that the blocking effect of BeKm-1 on
M-like current can be fully accounted for by inhibition of mERG1
channels in NG108-15 cells. Thus, BeKm-1 is a highly specific inhibitor
of ERG channels.
![]() |
ACKNOWLEDGEMENTS |
---|
Enterokinase was kindly provided by Dr. A. Mikhaylova. cDNA encoding hERG1 was a generous gift from G. Robertson. cDNAs encoding KCNQ2, KCNQ3, and KCNQ4 were kindly provided by Dr. Thomas Jentsch. cDNA encoding rELK1 was generously provided by Dr. Birgit Engeland, and cDNA for the hEAG was kindly provided by Dr. Jacqueline Fischer-Lougheed. KCNQ1 was from Dr. Jacques Barhanin. We thank Dr. E. D. Nosyreva for assistance in the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Russian Foundation of Fundamental Research.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/EMBL Data Bank with accession number(s) AF276623.
§ To whom correspondence should be addressed. Tel.: 7-095-3364022; Fax: 7-095-3307301; E-mail: july@ibch.ru.
Supported by the Danish Heart Foundation.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M005973200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EAG, ether-a-go-go gene; ERG, EAG-related; ELK, EAG-like; KCNQ, KQT-like K+ channel; BK, IK, SK, high-, intermediate-, small-conductance Ca2+-activated K+ channels; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; ORF, open reading frame; bp, base pair(s); HPLC, high performance liquid chromatography; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Filippov, A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280[CrossRef][Medline] [Order article via Infotrieve] |
2. | Christie, M. J. (1995) Clin. Exp. Pharmacol. Physiol. 22, 944-951[Medline] [Order article via Infotrieve] |
3. |
Coetzee, W. A.,
Amarillo, Y.,
Chiu, J.,
Chow, A.,
Lau, D.,
McCormack, T.,
Moreno, H.,
Nadal, M. S.,
Ozaita,
Pountney, A.,
Saganich, M.,
Vega-Saenz de Miera, E.,
and Rudy, B.
(1999)
Ann. N. Y. Acad. Sci.
868,
233-285 |
4. | Rowan, E. G., and Harvey, A. L. (1996) Braz. J. Med. Biol. Res. 29, 1765-1780[Medline] [Order article via Infotrieve] |
5. | Miller, C. (1995) Neuron 15, 5-10[Medline] [Order article via Infotrieve] |
6. | Harvey, A. L., Vatanpour, H., Rowan, E. G., Pinkasfeld, S., Vita, C., Menez, A., and Martineauclaire, M. F. (1995) Toxicon 33, 425-436[CrossRef][Medline] [Order article via Infotrieve] |
7. | Olamendi-Portugal, T., Gomez-Lagunas, F., Gurrola, G., and Possani, L. D. (1996) Biochem. J. 315, 977-981[Medline] [Order article via Infotrieve] |
8. | Kharrat, R., Marbouk, K., Crest, M., Darbon, H., Oughideni, R., Martineauclaire, M. F., Jacquet, G., Elayeb, M., Vanrietschoten, J., Rochat, H., and Sabatier, J. M. (1996) Eur. J. Biochem. 242, 491-498[Abstract] |
9. | MacKinnon, R., and Miller, C. (1989) Science 245, 1382-1385[Medline] [Order article via Infotrieve] |
10. | MacKinnon, R., Heginbotham, L., and Abramson, T. (1990) Neuron 5, 767-771[Medline] [Order article via Infotrieve] |
11. | MacKinnon, R. (1991) Nature 350, 232-235[CrossRef][Medline] [Order article via Infotrieve] |
12. | Hidalgo, P., and MacKinnon, R. (1995) Science 268, 307-310[Medline] [Order article via Infotrieve] |
13. | Naranjo, D., and Miller, C. (1996) Neuron 16, 123-130[Medline] [Order article via Infotrieve] |
14. |
Aiyar, J.,
Rizzi, J. P.,
Guttman, G. A.,
and Chandy, K. G.
(1996)
J. Biol. Chem.
271,
31013-31016 |
15. | Ranganathan, R., Lewis, J. H., and MacKinnon, R. (1996) Neuron 16, 131-139[Medline] [Order article via Infotrieve] |
16. | Lu, Z., and MacKinnon, R. (1997) Biochemistry 36, 6936-6940[CrossRef][Medline] [Order article via Infotrieve] |
17. | Brown, D. A., and Adams, P. R. (1980) Nature 283, 673-676[Medline] [Order article via Infotrieve] |
18. | Stansfeld, C., Ludwig, J., Roeper, J., Weseloh, R., Brown, D., and Pongs, O. (1997) Trends Neurosci. 20, 13-14[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Wang, H. S.,
Pan, Z.,
Shi, W.,
Brown, B. S.,
Wymore, R. S.,
Cohen, I. S.,
Dixon, J. E.,
and McKinnon, D.
(1998)
Science
282,
1890-1893 |
20. |
Selyanko, A. A.,
Hadley, J. K.,
Wood, I. C.,
Abogadie, F. C.,
Delmas, P.,
Buckley, N. J.,
London, B.,
and Brown, D. A.
(1999)
J. Neurosci.
19,
7742-7756 |
21. |
Meves, H.,
Schwarz, J. R.,
and Wulfsen, I.
(1999)
Br. J. Pharmacol.
127,
1213-1223 |
22. | Kawasaki, I., and Itano, H. A. (1972) Anal. Biochem. 48, 546-556[Medline] [Order article via Infotrieve] |
23. | Kiyatkin, N., Dulubova, I., and Grishin, E. (1993) Eur. J. Biochem. 213, 121-127[Abstract] |
24. | Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1413[Abstract] |
25. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
26. | Sanger, F., Nisclen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
27. | Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Science 243, 217-220[Medline] [Order article via Infotrieve] |
28. | Park, C.-S., and Miller, C. (1992) Neuron 9, 307-313[Medline] [Order article via Infotrieve] |
29. | Boyer, H. W., and Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, 459-472[Medline] [Order article via Infotrieve] |
30. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
31. | Mihailova, A. G., and Rumsh, L. D. (1999) FEBS Lett. 442, 226-230[CrossRef][Medline] [Order article via Infotrieve] |
32. | Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[Medline] [Order article via Infotrieve] |
33. | Robbins, J., Trouslard, J., Marsh, S. J., and Brown, D. A. (1992) J. Physiol. 451, 159-185[Abstract] |
34. | Adams, P. R., Brown, D. A., and Constanti, A. (1982) J. Physiol. 332, 223-262[Medline] [Order article via Infotrieve] |
35. | Brown, D. A., and Higashida, H. (1988) J. Physiol. 397, 167-184[Abstract] |
36. | Jensen, B. S., Strøbæk, D., Christophersen, P., Jørgensen, T. D., Hansen, C., Silahtaroglu, A., Olesen, S.-P., and Ahring, P. K. (1998) Am. J. Physiol. 275, 848-856 |
37. |
Strøbæk, D.,
Jørgensen, T. D.,
Christophersen, P.,
Ahring, P. K.,
and Olesen, S.-P.
(2000)
Br. J. Pharmacol.
129,
991-999 |
38. | Strøbæk, D., Christophersen, P., Holm, N. R., Moldt, P., Ahring, P. K., Johansen, T. E., and Olesen, S.-P. (1996) Neuropharmacology 35, 903-914[CrossRef][Medline] [Order article via Infotrieve] |
39. | Legros, C., Bougis, P. E., and Martin-Eauclaire, M. F. (1997) FEBS Lett. 402, 45-49[CrossRef][Medline] [Order article via Infotrieve] |
40. | Legros, C., Oughuideni, R., Darbon, H., Rochat, H., Bougis, P. E., and Martin-Eauclaire, M. F. (1996) FEBS Lett. 390, 81-84[CrossRef][Medline] [Order article via Infotrieve] |
41. | Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H., Timothy, K. W., Keating, M. T., and Goldstein, S. A. N. (1999) Cell 97, 175-187[Medline] [Order article via Infotrieve] |
42. |
Stansfeld, C. E.,
Roper, J.,
Ludwig, J.,
Weseloh, R. M.,
Marsh, S. J.,
Brown, D. A.,
and Pongs, O.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9910-9914 |
43. |
Engeland, B.,
Neu, A.,
Ludwig, J.,
Roeper, J.,
and Pongs, O.
(1998)
J. Physiol.
513,
647-654 |
44. |
Gurrola, G. B.,
Rosati, B.,
Rocchetti, M.,
Pimienta, G.,
Zaza, A.,
Arcangeli, A.,
Olivotto, M.,
Possani, L. D.,
and Wanke, E.
(1999)
FASEB. J.
13,
953-962 |
45. | Bontems, F., Roumestand, C., Boyot, P., Gilquin, B., Doljansky, Y., Menez, A., and Toma, F. (1991) Eur. J. Biochem. 196, 19-28[Abstract] |
46. | Johnson, B. A., and Sugg, E. E. (1992) Biochemistry 31, 8151-8159[Medline] [Order article via Infotrieve] |
47. | Johnson, B. A., Stevens, S. P., and Williamson, J. M. (1994) Biochemistry 33, 15061-15070[Medline] [Order article via Infotrieve] |
48. | Fernandez, I., Romi, R., Szendeffy, S., Martineauclaire, M. F., Rochat, H., Vanrietschoten, J., Pons, M., and Giralt, E. (1994) Biochemistry 33, 14256-14263[Medline] [Order article via Infotrieve] |
49. | Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) Trends Pharmacol. Sci. 20, 444-447[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Smith, C.,
Phillips, M.,
and Miller, C.
(1986)
J. Biol. Chem.
261,
14607-14613 |
51. | MacKinnon, R., Reinhart, P. H., and White, M. M. (1988) Neuron 1, 997-1001[Medline] [Order article via Infotrieve] |
52. | Goldstein, S. A. N., Pheasant, D. J., and Miller, C. (1994) Neuron 12, 1377-1388[Medline] [Order article via Infotrieve] |
53. | Olamendi-Portugal, T., Gomez-Lagunas, F., Gurrola, G. B., and Possani, L. D. (1998) Toxicon 36, 759-770[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Tremeau, O.,
Lemaire, C.,
Drevet, P.,
Pinkasfeld, S.,
Ducancel, F.,
Boulain, J. C.,
and Menez, A.
(1995)
J. Biol. Chem.
270,
9362-9369 |
55. | Bouhaouala-Zahar, B., Ducancel, F., Zenouaki, I., Ben Khalifa, R., Borchani, L., Pelhate, M., Boulainm, J. C., El Ayeb, M., Menez, A., and Karoui, H. (1996) Eur. J. Biochem. 238, 653-660[Abstract] |
56. | Park, C.-S., Hausdorff, S. F., and Miller, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2046-2050[Abstract] |
57. |
Garcia-Calvo, M.,
Leonard, R. J.,
Novick, J.,
Stevens, S. P.,
Schmalhofer, W.,
Kaczorowski, G. J.,
and Garcia, M. L.
(1993)
J. Biol. Chem.
268,
18866-18874 |
58. | Legros, C., Feyfant, E., Sampieri, F., Rochat, H., Bougis, P. E., and Martin-Eauclaire, M. F. (1997) FEBS Lett. 417, 123-129[CrossRef][Medline] [Order article via Infotrieve] |