SO-3, a new O-superfamily conopeptide derived from Conus striatus, selectively inhibits N-type calcium currents in cultured hippocampal neurons.

Abstract

Whole-cell currents in cultured hippocampal neurons were recorded to investigate the effects of SO-3, a new O-superfamily conopeptide derived from Conus striatus, on voltage-sensitive channels. SO-3 had no effect on voltage-sensitive sodium currents, delayed rectifier potassium currents, and transient outward potassium currents. Similar to the selective N-type calcium channel blocker omega-conotoxin MVIIA (MVIIA), SO-3 could concentration-dependently inhibit the high voltage-activated (HVA) calcium currents (I(Ca)). MVIIA(3 microM), 10 microM nimodipine, and 0.5 microM omega-agatoxin IVA (Aga) could selectively block the N-, L-, and P/Q-type I(Ca), which contributed approximately 32, approximately 38, and approximately 21% of the HVA currents in hippocampal neurons, respectively. About 31% of the total HVA currents were inhibited by 3 microM SO-3. SO-3 (3 microM) and 3 microM MVIIA inhibited the overlapping components of HVA currents, whereas no overlapping component was inhibited by 3 microM SO-3 and 10 microM nimodipine, or by 3 microM SO-3 and 0.5 microM Aga. Also, 3 microM SO-3 had no effect on R-type currents. SO-3 had less inhibitory effects on non-N-type HVA currents than MVIIA at higher concentrations (30 and 100 microM). The inhibitory effects of SO-3 and MVIIA on HVA currents were almost fully reversible. However, the recovery from block by MVIIA was more rapid than recovery from block by SO-3. It is concluded that SO-3 is a new omega-conotoxin selectively targeting N-type voltage-sensitive calcium channels. Considering the significance of N-type calcium channels for pain transduction, SO-3 may have therapeutic potential as a novel analgesic agent.

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SO-3, a new O-superfamily conopeptide derived from Conus
striatus, selectively inhibits N-type calcium currents in cultured
hippocampal neurons
1Lei Wen,1Sheng Yang,1Haifa Qiao,1Zhenwei Liu,1Wenxia Zhou, *,1Yongxiang Zhang
&2Peitang Huang
1Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Haidian District, Beijing 100850, China and
2Beijing Institute of Biotechnology, Beijing, China
1
SO-3, a new O-superfamily conopeptide derived from Conus striatus, on voltage-sensitive channels.
2SO-3 had no effect on voltage-sensitive sodium currents, delayed rectifier potassium currents, and
transient outward potassium currents.
3 Similar to the selective N-type calcium channel blocker o-conotoxin MVIIA (MVIIA), SO-3 could
concentration-dependently inhibit the high voltage-activated (HVA) calcium currents (ICa).
4 MVIIA(3mM), 10mM nimodipine, and 0.5mM o-agatoxin IVA (Aga) could selectively block the
N-, L-, and P/Q-type ICa, which contributed B32, B38, and B21% of the HVA currents in
hippocampal neurons, respectively. About 31% of the total HVA currents were inhibited by 3mM
SO-3. SO-3 (3mM) and 3mM MVIIA inhibited the overlapping components of HVA currents, whereas
no overlapping component was inhibited by 3mM SO-3 and 10mM nimodipine, or by 3mM SO-3 and
0.5mM Aga. Also, 3mM SO-3 had no effect on R-type currents.
5SO-3 had less inhibitory effects on non-N-type HVA currents than MVIIA at higher
concentrations (30 and 100mM).
6 The inhibitory effects of SO-3 and MVIIA on HVA currents were almost fully reversible.
However, the recovery from block by MVIIA was more rapid than recovery from block by SO-3.
7 It is concluded that SO-3 is a new o-conotoxin selectively targeting N-type voltage-sensitive
calcium channels. Considering the significance of N-type calcium channels for pain transduction, SO-3
may have therapeutic potential as a novel analgesic agent.
British Journal of Pharmacology (2005) 145, 728–739. doi:10.1038/sj.bjp.0706223;
published online 9 May 2005
SO-3; o-conotoxins; voltage-sensitive calcium channels; voltage-sensitive potassium channels; voltage-sensitive
sodium channels; N-type calcium channel blockers; pain; hippocampal neurons; patch-clamp techniques
Whole-cell currents in cultured hippocampal neurons were recorded to investigate the effects of
Keywords:
Abbreviations:Aga, o-agatoxin IVA; CVID, o-conotoxin CVID; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N,N0,N0-
tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HVA, high voltage-activated; ICa,
calcium currents; IK(A), transient outward potassium currents; IK(DR), delayed rectifier potassium currents; INa,
sodium currents; I–V, current–voltage; MVIIA, o-conotoxin MVIIA; TEA-Cl, tetraethylammonium chloride;
TTX, tetrodotoxin; Vh, holding potential; VSCCs, voltage-sensitive calcium channels; VSPCs, voltage-sensitive
potassium channels; VSSCs, voltage-sensitive sodium channels
Introduction
Conopeptides are small, structured peptide toxins secreted by
the venomous marine snails of the genus Conus for prey
capture, defense, and competitor deterrence. It is now clear that
in the venoms of living species in the genus Conus there are
about 50,000 different conotoxins, most of which are 12–30
amino acids in length and contain two to four disulfide bridges,
and can selectively target a specific voltage-gated ion channel,
ligand-gated ion channel, or G-protein-coupled receptor. These
potential pharmaceutical benefits have led to the use of certain
conotoxins as tools for ion channel research as well as for the
treatment and diagnosis of neurological diseases (Nielsen et al.,
2000; Olivera & Cruz, 2001; Terlau & Olivera, 2004).
Conotoxins are defined to a number of structural classes
according to the characteristic arrangement of Cys-residues.
The O-superfamily, which contains four-loop, 6-Cys peptides,
comprises o-conotoxins (voltage-sensitive calcium channel
(VSCC) blockers), k-conotoxins (voltage-sensitive potassium
channel (VSPC) blockers), mO-conotoxins (voltage-sensitive
sodium channel (VSSC) blockers), and d-conotoxins (inhibi-
tors of the fast inactivation of VSSCs). These four classes of
conotoxins are unusually hydrophobic peptides with the same
cysteine framework C–C–CC–C–C, giving these peptides a
similar inhibitory cysteine knot (ICK) motif (Table 1) (Nielsen
et al., 2000; Olivera & Cruz, 2001; Terlau & Olivera, 2004).
The individual peptides in one conotoxin family may
selectively target a diverse set of different molecular isoforms
within the same ion channel family. For example, o-conotoxin
*Author for correspondence; E-mail: zhangyx@nic.bmi.ac.cn
British Journal of Pharmacology (2005) 145, 728–739
& 2005 Nature Publishing GroupAll rights reserved 0007–1188/05 $30.00
www.nature.com/bjp

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MVIIA and MVIIC, both derived from Conus magus, have high
identity in sequence, but quite different selectivity: MVIIA is
highly selective for N-type VSCCs, whereas MVIIC is more
selective for the P/Q-type (Nielsen et al., 1999a). Among the o-
conotoxins CVIA–CVID, all identified from C. catus, CVIA and
CVID are selective towards the N-type VSCCs, while CVIB and
CVIC show no difference between N- and P/Q-type VSCCs
(Lewis et al., 2000; Nielsen et al., 2000; Adams et al., 2003). On
the other hand, peptides derived from different cone snail species,
although their conopeptide sequences vary largely except the
cysteine framework, may act on the same ion channel subtype, as
both o-conotoxin MVIIA and GVIA can block the same N-type
VSCCs (Olivera & Cruz, 2001; Terlau & Olivera, 2004) (Table 1).
The venoms of each species always contain a unique array of
more than 100 peptides, which are used by the genus Conus for
divergent biotic purposes. For this reason, corresponding
venom components with divergent pharmacological properties
in one particular Conus species are expected. In searching for
new conotoxins with some therapeutic potential, more than 25
species of Conus from the South China Sea have been collected.
Also, by gene screening, we identified a new conopeptide termed
SO-3 from a fish-eating snail, C. striatus (Lu et al., 1999). SO-3
contains 25 amino-acid residues and the same cysteine frame-
work as O-superfamily conotoxins. Previously, three O-super-
family conopeptides, including two o-conotoxins (SVIA and
SVIB) and one d-conotoxin (SVIE), have been isolated from the
venom of C. striatus biochemically (Table 1) (Ramilo et al.,
1992; Bulaj et al., 2001). SO-3 shows 56% sequence identity
with SVIB, a P/Q-type VSCCs blocker, and 72% with MVIIA,
an N-type VSCC blocker. In addition, it has the same positive
charges as SVIB and MVIIA (Table 1). Further bioactivity
evaluation on the synthetic SO-3 showed that SO-3 is a potent
analgesic agent with effects similar to MVIIA (Dai et al., 2003).
However, the ion channel target of SO-3 is still unclear. Here,
we observed the effects of SO-3 on voltage-sensitive ion currents
in primary cultured hippocampal neurons. The results indicated
that SO-3 is a selective N-type VSCC blocker.
Methods
Cell culture
Primary cultures of hippocampal cells were established using a
modification of the previous procedure (Banker & Cowan,
1979). Wistar rats at postnatal day 1 were deeply anesthetized
with ether and decapitated, in accordance with the guideline of
the Beijing Institutes for Biological Sciences Animal Research
Advisory Committee. The hippocampi were dissected in ice-
cold Ca2þ- and Mg2þ-free PBS, incubated at 371C with
0.125% trypsin (type XI; Sigma Co., Ltd, St Louis, MO,
U.S.A.) for 20min, and then triturated to dissociate cells. After
being washed, the single cell suspension was diluted with
Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 10% horse serum (HS),
100Uml?1penicillin, and 100mgml?1streptomycin. Cells were
plated in 35mm plastic culture dishes (Nunc, Denmark) pre-
coated with poly-L-lysine (75mgml?1) at a density of
5.0?105cells per dish, and cultured at 371C in a humidified
atmosphere containing 5% CO2 and 95% air. After 24-h
incubation, the culture medium was changed to 2ml of
DMEM containing 10% FBS, 2mM glutamine, 1% N2,
100Uml?1penicillin, and 100mgml?1streptomycin. The
proliferation of non-neuronal cells was controlled by treating
with 3mM cytosine arabinofuranoside (c-Ara) for 48h at the
second day in culture. Subsequently, half of the medium was
replaced twice a week. All experiments were performed on cells
aging 10–12 days in vitro. c-Ara was purchased from Sigma
Co., Ltd, and other reagents including DMEM, FBS, HS, and
N2were obtained from GIBCO Life Technologies (Rockville,
MD, U.S.A.).
Recording solutions
The extracellular solution for whole-cell recordings contained
(in mM): NaCl 140, KCl 5, MgCl21, CaCl22, Dextrose 10,
Table 1Selected O-superfamily conopeptides targeting different voltage-sensitive ion channels
Peptide Conus SpecieSequencea
Targeted ion channelb
Reference
C------C------CC---C------C∆
loop 1 loop 2
CKSOGSSCSOTSYNCCR-SCNOYTKRCY* (4)
CKGKGAKCSRLMYDCCTGSC–RSGKC* (5)
CKGKGAPCRKTMYDCCSGSCGRR-GKC* (6)
CKSTGASCRRTSYDCCTGSC–RSGRC* (4)
CKGKGASCRKTMYDCCRGSC–RSGRC* (6)
CKGKGQSCSKLMYDCCTGSCSRR-GKC* (5)
CKSKGAKCSKLMYDCCSGSCSGTVGRC* (4)
CRSSGSPCGVTS-ICC-GRC–YRGKCT* (4)
CKLKGQSCRKTSYDCCSGSCG-RSGKC* (5)
CKAAGKPCSRIAYNCCTGSC–RSGKC* (5)
DGCSSGGTFCGIHOGLCCSEFCFLWCITFID
ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV
ACSKKWEYCIVPILGFVYCCPGLICGPFVCV
CRIONQKCFQHLDDCCSRKCNRFNKCV
loop 3loop 4
o-GVIA
o-MVIIA
o-MVIIC
o-CVIA
o-CVIB
o-CVIC
o-CVID
o-SVIA
o-SVIB
SO-3c
d-SVIE
mO-MrVIA
mO-MrVIB
k-PVIIA
C. geographus
C. magus
C. magus
C. catus
C. catus
C. catus
C. catus
C. striatus
C. striatus
C. striatus
C. striatus
C. marmoreus
C. marmoreus
C. purpurascens
VSCCs, N-type
VSCCs, N-type
VSCCs, P/Q/N-type
VSCCs, N-type
VSCCs, N/P/Q-type
VSCCs, N/P/Q-type
VSCCs, N-type
poor activity on VSCCs
VSCCs, P/Q-type
?
VSSCs
VSSCs
VSSCs
VSPCs
Olivera et al. (1984)
Olivera & Cruz (2001)
Hillyard et al. (1992)
Lewis et al. (2000)
Lewis et al. (2000)
Lewis et al. (2000)
Lewis et al. (2000)
Ramilo et al. (1992)
Ramilo et al. (1992)
Bulaj et al. (2001)
McIntosh et al. (1995)
McIntosh et al. (1995)
Shon et al. (1998)
aAll o-conotoxins are C-terminally amidated (*), each net positive charge is indicated in the braces after the sequence of the corresponding
o-conotoxin. The disulfide bridges motif of o-conotoxins and its four loops (D) are also displayed above the sequences.
bTargeted ion channels are determined from radioligand binding and/or electrophysiology experiments.
cGenBank accession number of the SO-3 cDNA sequence is AF46350.
L. Wen et al
New N-type Ca channel blocker from Conus striatus
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British Journal of Pharmacology vol 145 (6)

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HEPES 10. The pH for extracellular solution was adjusted to
7.4 with NaOH and the osmolarity was adjusted to 320mOsm
with sucrose. For Kþcurrent recording, the extracellular
solution was supplemented with 1mM tetrodotoxin (TTX) to
block Naþchannels, and 0.4mM CdCl2 to block all Ca2þ
channels and Ca2þ-activated Kþchannels. For Naþcurrent
recording, the same extracellular solution was used, except
TTX was replaced with 5mM tetraethylammonium chloride
(TEA-Cl) and 2mM 4-aminopyridine (4-AP) to block the
delayed rectified Kþchannels and transient outward Kþ
channels, respectively, For Ca2þcurrent recording, 1mM TTX,
5mM TEA-Cl, and 2mM 4-AP were added to the extracellular
solution to block Naþ
channels and Kþ
intracellular pipette solution used for Kþcurrent recording
contained (in mM): KCl 140, HEPES 10, EGTA 10, and ATP-
Na22, and the pH was adjusted to 7.2 by KOH. For Naþand
Ca2þcurrent recording, the solution contained (in mM): CsCl
140, HEPES 10, EGTA 10, TEA-Cl 5, and ATP-Na2,2, and the
pH was adjusted to 7.2 by CsOH. The osmolarity of the pipette
solutions was adjusted to 320mOsm. TEA-Cl, 4-AP, TTX,
CdCl2, and ATP-Na2were purchased from Sigma Co., Ltd.
channels. The
Electrophysiology recordings
Patch pipettes (with resistances of 2–4MO after being filled
with intracellular solutions) were prepared using a two-stage
microelectrode puller (PC-10; Narishige, Tokyo, Japan) and
fire-polished using a MF-830 microforge (Narishige) just
before recording. All experiments were carried out under
room temperature (20–221C). Voltage-clamp recordings were
performed in the whole-cell patch-clamp configuration accord-
ing to standard patch-clamp methods (Hamill et al., 1981)
using an Axopatch 200B patch-clamp amplifier (Axon Instru-
ments, Foster City, CA, U.S.A.). Data were sampled at 5kHz
and filtered at 2kHz (?3dB, four-pole Bessel filter) on-line by
using the pCLAMP 8.2 software (Axon Instruments, Foster
City, CA, U.S.A.). Series resistance was compensated (480%)
in all experiments, and leak subtraction was carried out using a
P/?4 protocol online. For all experiments, as soon as the
whole-cell configuration had been acquired, current was
allowed to stabilize for approximately 5min before the
experimental voltage command was administered. Only those
recordings with stable holding currents and access resistance
were accepted. Cells showing current rundown at a rate of
currents more than 1%min?1in the absence of treatments
were discarded. All calcium currents (ICa) were normalized for
differences in cell sizes by transformation to current density
(pApF?1; dividing each current (Ica) measure by the whole-cell
membrane capacitance for that cell).
Drugs and drug application
The synthesis, folding, and purification of SO-3 were
performed in Beijing Institute of Biotechnology, China as
described in Dai et al. (2003). Using the designated conditions,
the folding peptide of SO-3 was obtained in greater than 98%
purity after two steps of HPLC purification. The final peptide
content in lyophilized powder was approximately 79%. Using
MALDI-TOF, the molecular mass (single isotope) of SO-3 was
2560.1Da, in agreement with the calculated molecular mass of
2561.1Da. Nimodipine, o-conotoxin MVIIA (MVIIA), and
o-agatoxin IVA (Aga) were purchased from Sigma Co., Ltd.
Nimodipine was stored in opaque containers at ?201C as a
10mM stock solution in 100% ethanol. SO-3, MVIIA, and
Aga were dissolved in extracelllular solutions to make 1mM
stock solutions that were aliquot and stored at ?201C. Each
stock solution was diluted to the test concentration with
extracelular solution. Bovine serum albumin (BSA (1mgml?1);
Sigma) was present in all final extracelular solutions to prevent
the nonspecific binding of peptides. To control for vehicle
effects, 0.1% (vv?1) ethanol was present in the extracelular
solutions throughout the experiments when nimodipine would
be applied, and experiments were performed under restricted
light conditions. During the experiments, cells were rinsed
twice with extracellular solution, then continuously perfused
with the test concentration of drug(s) by using a gravity-fed
bath perfusion system. The flow rate was approximately
5?10?3mls?1. The distance of the flow pipette from the cell
was approximately 150mm and the pipette tip diameter was
about 300mm. The total volume of the extracellular solution
was kept in 1.5ml during the experiments.
Data analysis
The concentration–response curves for the effects of SO-3 and
MVIIA on the high voltage-activated (HVA) ICawere fitted
according to the logistic equation
EðCÞ=Emax¼ 1=½1 þ ðC=EC50ÞnH?
where C is the concentration of SO-3 or MVIIA, E(C) is the
effect evoked by SO-3 or MVIIA at concentration C, Emax
denotes the maximal possible effect, EC50is the concentration
at which half-maximal effect is obtained. nH is the Hill
coefficient.Origin 6.0 (OriginLab,
U.S.A.) software was used to perform nonlinear fit of data.
SPSS 12.0 software (SPSS Inc., Chicago, IL, U.S.A.) was used
for statistical analysis. All data are expressed as mean7s.e.m.,
and Po0.05 indicates statistically significant.
Northampton,MA,
Results
No effect of SO-3 on voltage-sensitive sodium currents
(INa)
Hippocampal neurons were clamped at a holding potential
(Vh) of ?80mV, and depolarized to ?40mV at 10s intervals to
activate inward Naþcurrents which were blocked by 1mM
TTX completely and reversibly (data were not shown). After a
3-min application of 1, 10, and even 100mM SO-3, the
INaamplitudes were not affected. The current–voltage (I–V)
curves remained unaffected after application of 100mM SO-3.
These observations indicated that SO-3 had no effect on INa
(Figure 1).
No effect of SO-3 on delayed rectifier potassium currents
(IK(DR)) and transient outward potassium currents
(IK(A))
IK(DR) and IK(A) were isolated using the electrophysiological
and pharmacological methods (Yang et al., 2001). The
experimental pulse protocols and subtraction procedure are
shown in Figure 2a. Currents at the end of the depolarizing
pulse (148ms) were referred to as IK(DR). The peak currents of
730
L. Wen et al
New N-type Ca channel blocker from Conus striatus
British Journal of Pharmacology vol 145 (6)

Page 4

the subtracted traces were referred to as IK(A). As Figure 2b
and c showed, no significant effect on IK(DR)and IK(A)was
observed after exposure to 100mM SO-3.
Concentration-dependent inhibition of SO-3 on HVA ICa
HVA ICa were evoked by 150ms depolarizing voltage step
commands from a Vh of ?80mV in primary cultured
hippocampal neurons. Under the present experiments proce-
dure, the inward ICa reached maximum amplitude when
depolarizing to 0mV. Figure 3 indicated a concentration-
dependent inhibition of SO-3 on HVA ICa, and these inhibitory
effects were similar to that of MVIIA, a determined N-type
calcium channel blocker. In most cases, the inhibitory effects
of a given concentration of peptides occurred within 10–20s
application and reached a stable maximum after 30–50s. For
both SO-3 and MVIIA, there was a plateau of inhibition at
1–3mM. The calculated EC50’s for SO-3 and MVIIA were
0.16 and 0.20mM, respectively.
Inhibitory effects of SO-3 on I–V relationships of ICa
To identify whether the inhibition of ICacaused by SO-3 in
cultured hippocampal neurons is voltage-dependent, the I–V
relationships in the presence or absence of SO-3 were studied
(Figure 4). These results confirmed the concentration-depen-
dent inhibition on ICaafter 2-min exposure to 0.03, 0.3, and
3mM SO-3. The inhibitory effects of SO-3 on ICawere obvious
at the potentials between –20 and þ40mV, and no obvious ICa
was inhibited below the potential of –30mV. These results
indicated that SO-3 had inhibitory effects on HVA ICa, other
than on low voltage-activated (LVA), that is, T-type currents.
I–V relationships also showed no significant change of the
threshold of activation of ICaand the reversal potential after
application of SO-3. In order to illustrate the effects of SO-3
more clearly, the I–V curve of the 3mM SO-3-sensitive currents
was obtained by subtracting the currents in the presence of
3mM SO-3 from the currents in the absence of 3mM SO-3. The
I–V curve of the subtracted 3mM SO-3-sensitive currents was
very similar to the subtracted 3mM MVIIA-sensitive currents,
indicated that, at the concentration of 3mM, SO-3 and MVIIA
had similar effects on I–V relationships of ICa.
Selectivity of SO-3 on VSCC subtype
Previous experiments have proved the inhibitory effects of SO-
3 on HVA ICa; here, we further examined whether HVA ICa
inhibited by SO-3 is VSCC-subtype selective. At least four
distinct types of HVA VSCCs (L-, N-,P/Q-, and R-type) are
expressed in cultured hippocampal neurons and sensitive to
different blockers (Currie & Fox, 1997; Nakashima et al.,
1998; Newcomb et al., 1998; Blalock et al., 1999; Scamps et al.,
2000). In our studies, SO-3 and one of three distinct VSCC
blockers (MVIIA, nimodipine or Aga) were additionally
applied to cultured hippocampal neurons in three separate
experiments with drug presentation order reversed (Figures
5–7). According to previous reports (Nakashima et al., 1998;
Blalock et al., 1999; Scamps et al., 2000), 10mM nimodipine,
3mM MVIIA, and 0.5mM Aga were used to define L-, N-, and
P/Q-type currents in these experiments, respectively. SO-3
(3mM) was also used. Typically, SO-3, MVIIA, and Aga
required 30–50s exposure to give stable inhibition, whereas
nimodipine acted more rapidly (B20–30s).
As Figure 5 showed, after application of 3mM SO-3 or
MVIIA, the inhibition amounts of HVA ICawere similar (B31
and B32%, respectively), and no further inhibition was
observed after additional application of the other drug. The
remained HVA ICa could be blocked almost completely by
0.4mM CdCl2within 20s (data were not shown). These results
suggested that SO-3 and MVIIA inhibited the overlapping
components of HVA ICa in cultured hippocampal neurons
(two-way ANOVA on repeated measures, Po0.001 for
presentation order), and there was no significant difference
between the effects of SO-3 and MVIIA (P¼0.872 for drug
type). Therefore, these two drugs may share a common site of
inhibition in this preparation. Unlike the experiments de-
scribed above, sequential addition of SO-3 and nimodipine,
and SO-3 and Aga, both produced additive inhibition on HVA
06
0.0
0.4
0.8
1.2
0
21
1
2
− 40 mV
− 80 mV
2 ms
500 pA
NormalizedINa
Time (min)
24810
1 ? ?M10 ? ?M 100 ? ?M SO-3
Control
100 ? ?M SO-3
Membrane Potential (mV)
-80-60-40-20 20 40
I Na Amplitude (nA)
0.0
-0.4
-0.8
-1.2
-1.6
a
b
Figure 1
cultured hippocampal neurons. (a) The time course of the effects of
1, 10 and 100mM SO-3 on peak INa. Currents were evoked by 20ms
depolarizing voltage step commands from a Vhof ?80 to ?40mV at
10s intervals. Peak INaamplitudes were normalized to the values in
the absence of SO-3 and plotted against exposure time. Data are
shown as mean7s.e.m., n¼13. Drugs were applied to the cell as
indicated by the horizontal bar. Inset: Pulse paradigm and the
representative current traces at the time point 1 (black) and 2 (gray).
(b) I–V relationship of peak INabefore and after 2-min exposure to
100mM SO-3. Each point represents the mean of eight cells. INa’s
were evoked by a series of 20ms depolarizing steps from a Vhof ?80
to þ40mV, with 10mV increments and 2s intervals.
No effect of SO-3 on voltage-sensitive INa in primary
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British Journal of Pharmacology vol 145 (6)

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ICa; 3mM SO-3-, 10mM nimodipine-, and 0.5mM Aga-sensitive
currents contributed B31, B38, and B21% of the total HVA
ICa, respectively, regardless of the presentation order of these
blockers (for the presentation order, for SO-3/nimodipine pair,
P¼0.682; for SO-3/Aga pair, P¼0.544), and there were
significant differences between the effects of SO-3 and
nimodipine(P¼0.008),
(P¼0.001) (Figures 6 and 7). These results indicated that no
overlapping component of HVA ICawas inhibited by SO-3 and
nimodipine, or by SO-3 and Aga, and suggested that SO-3 did
not inhibit L- and P/Q-type currents at the concentration
tested.
The effects of SO-3 on R-type HVA ICa were further
examined. L-, N-, and P/Q-type currents were blocked by
10mM nimodipine, 3mM MVIIA, and 0.5mM Aga, and the
isolated R-type currents contributed B12% of the total
HVA ICa. After exposure to 3mM SO-3, the R-type currents
remained unaffected (Figure 8). These results also suggested
that SO-3 did not inhibit R-type currents at the concentra-
tion tested.
Several previous studies have demonstrated that o-con-
otoxin GVIA and MVIIA can block the heterologously
expressed L-, P/Q-, and R-type VSCCs at high concentrations
(Williams et al., 1992b; Zhang et al., 1993; Feng et al., 2003).
We also observed the effects of the higher concentrations of
andbetweenSO-3 andAga
SO-3 on non-N-type HVA ICa in cultured hippocampal
neurons. As Figure 9 showed, 3mM SO-3 and MVIIA were
first applied to block the N-type ICa, the remaining HVA ICa’s
were both further inhibited by additional application of 30 and
100mM SO-3 and MVIIA. About 7 and 13% of the total HVA
ICawere further inhibited by 30 and 100mM SO-3, respectively,
and more fractions of HVA ICa(B15 and B37%, respectively)
were further inhibited by 30 and 100mM of MVIIA (unpaired
Student’s t-test, Po0.01). These results indicated that SO-3
had less inhibitory effects on non-N-type VSCCs than MVIIA
at high concentrations.
Reversibility of the inhibitory effects of SO-3 on HVA ICa
The different degree of recovery from the block effects of
several o-conotoxins, including GVIA, MVIIA, and CVID, on
VSCCs was reported previously (Williams et al., 1992a;
Kristipati et al., 1994; Lin et al., 1997; Stocker et al., 1997;
Feng et al., 2001; 2003; Liang & Elmslie, 2002; Mould et al.,
2004). Under the present experiments procedure, the inhibitory
effects of SO-3 and MVIIA on HVA ICawere both almost fully
reversible. However, the recovery from block by MVIIA was
more rapid than recovery from block by SO-3. The half-time
for recovery for SO-3 and MVIIA were 7.5270.56min
0 2040 60
0
1
2
3
4
0
1
2
3
4
5
+ +60 mV
−110 mV
400 ms
150 ms
150 ms
50 ms
450 ms
−50 mV
1 nA
50 ms
100 ? ?M SO-3
I K (DR) Amplitude (nA)
I K (A) Amplitude (nA)
Control
100 ? ?M SO-3
Control
-60 -40
Membrane Potential (mV)
-20
02040 60-60-40
Membrane Potential (mV)
-20
a
b
c
Figure 2
(IK) (left), IK(DR)(middle) and IK(A)(right). The upper inset shows the stimulus waveforms of IKand IK(DR), respectively. IKwere
evoked by 150ms depolarizing pulses from ?50 to þ60mV in 10mV steps following a hyperpolarizing prepulse of 450ms to
?110mV. IK(DR) were elicited using a similar protocol, except that for a 50ms interval at ?50mV was inserted after the
hyperpolarizing prepulse of 400ms to ?110mV. IK(A)were isolated by subtracting the trace of IK(DR)from IK. (b, c) I–V curves of
IK(DR)and IK(A), both n¼9. Currents were recorded before (black) and after (gray) 2-min exposure to 100mM SO-3.
No effect of SO-3 on voltage-sensitive potassium currents. (a) The example family traces of outward potassium currents
732
L. Wen et al
New N-type Ca channel blocker from Conus striatus
British Journal of Pharmacology vol 145 (6)