From the School of Biomedical Sciences and
¶ Institute for Molecular Bioscience, The University of
Queensland, Brisbane, Queensland 4072, Australia
Received for publication, September 30, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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Neurotransmitter release from preganglionic
parasympathetic neurons is resistant to inhibition by selective
antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this
study, the effects of different Venom of the predatory marine gastropods of the genus
Conus (cone snails) contain a unique array of peptides whose
pharmaceutical potential remains largely unexploited (1).
These peptides have been classified based on their
pharmacological target and structure (2, 3). One important class, the
Neurotransmitter release from preganglionic parasympathetic neurons has
been found to be resistant to inhibition by a range of selective
calcium channel antagonists of L-, N-, P/Q-, R-, and T-type calcium
channels (5-8). A recently discovered The orientation and nature of the residues in loop 2 of Submandibular Ganglia Preparation--
2-5-week-old rats were
anesthetized and killed by cervical fracture prior to the removal of
the submandibular ganglia in accordance with the guidelines of the
University of Queensland Animal Experimentation Committee. The
submandibular ganglia were removed as described previously (8, 16).
Individual preparations were pinned to the Sylgard (Dow Corning)
covered base of a 2-ml Perspex organ bath. Preparations were
continuously perfused at a rate of 2 ml/min with a Krebs phosphate
solution of the following composition (in mM): 118.4 NaCl, 25.0 NaHCO3, 1.13 NaH2PO4,
1.8 CaCl2, 4.7 KCl, 1.3 MgCl2, and 11.1 glucose, gassed with a mixture of 95% O2 and 5%
CO2 to pH 7.4, and maintained at 36-37 °C.
Intracellular Recordings and Analysis--
The lingual nerve was
field-stimulated by voltage pulses via bare platinum wires delivered
from a digital stimulator (Pulsar 7+, Frederick Haer & Company, Brunswick, ME) coupled to an optically isolated stimulation
unit (Model DS2, Digitimer Ltd., Welwyn Garden City, United Kingdom).
Intracellular recordings were made from individual ganglion cells using
glass microelectrodes filled with 5 M potassium acetate
(resistances 70-120 megohms). Conventional intracellular recording
techniques were used as described previously (17, 18). Membrane
potentials were recorded through a headstage connected to an
Axoclamp-2A amplifier (Axon Instruments Inc., Union City, CA) in bridge
mode and stored on a digital audio tape using a digital tape
recorder (DTR-1204, BioLogic Science Instruments, Claix, France).
Evoked events were digitized at 5-10 kHz and transferred to a computer
using an analogue-to-digital converter (Digidata 1200A interface) and
Axotape software (Axon Instruments Inc.). The amplitude, frequency,
rise time, and latency of evoked and spontaneous events were analyzed
using Axograph 2 (Axon Instruments Inc.). The mean resting membrane
potential of the submandibular ganglion neurons was Electrophysiological Recording of Recombinant Ca2+
Channel Currents in Xenopus Oocytes--
Capped RNA transcripts
encoding full-length rat Radioligand Binding--
Preparation of rat brain membrane and
[125I]GVIA and radioligand displacement protocols
were as described previously (9, 14, 15).
1H NMR Spectroscopy--
All NMR spectra were
recorded on a Bruker ARX 500 spectrometer equipped with a
z-gradient unit or on a Bruker DMX 750 spectrometer equipped
with a x,y,z-gradient unit. Peptide concentrations were ~2
mM. H Drugs--
Drugs were dissolved in the Krebs phosphate solution
perfusing the preparation, and the effects were evaluated after they reached equilibrium ( Characteristics of Excitatory Postsynaptic Potentials
(EPSPs)--
Stimulation of the lingual nerve with trains of stimuli
(0.1-10 Hz, 4-50 V, pulse-width 0.05-0.25 ms) evoked EPSPs, which could initiate action potentials in the cell bodies of the postsynaptic neurons of the rat submandibular ganglion. The postganglionic neurons
have been classified into three types by their responses to these
trains of stimuli (8). In this study, neurons in which supramaximal
stimulation of the preganglionic nerve fibers at 0.1 Hz either evoked a
suprathreshold EPSP and action potential in response to every stimulus
(strong input synapse, Effects of Calcium Channel Inhibitors on EPSPs--
The effect of CVID contrasts with the effects of 300 nM
Of the characterized
The effects of Radioligand Binding Studies--
The affinity of Effect of Three Dimensional Structures--
H Neurotransmitter release from the preganglionic cholinergic
neurons innervating the rat submandibular ganglia is resistant to
inhibition by commonly used selective antagonists of N-, L-, P/Q-, R-,
and T-type calcium channels (8, 17). The aim of this study was to
investigate the effects of the recently identified Loop 2 of The effects of the L-, N-, P/Q-, and R-type VSCC antagonists
(nifedipine, In an attempt to understand the structural basis of the selectivity
differences between In conclusion, CVID was found to be a potent inhibitor of the
pharmacologically distinct VSCC controlling a major component of
transmitter release in preganglionic cholinergic nerve terminals. CVID
may help to define the nature and role of this VSCC. Position 10 appears to play a key role in influencing -conotoxins from genus
Conus were investigated on current flow-through cloned
voltage-sensitive calcium channels expressed in Xenopus
oocytes and nerve-evoked transmitter release from the intact
preganglionic cholinergic nerves innervating the rat submandibular
ganglia. Our results indicate that
-conotoxin CVID from Conus
catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas
-conotoxin MVIIA had no effect.
-Conotoxin CVID and MVIIA
inhibited depolarization-activated Ba2+ currents recorded
from oocytes expressing N-type but not L- or R-type calcium channels.
High affinity inhibition of the CVID-sensitive calcium channel was
enhanced when position 10 of the
-conotoxin was occupied by the
smaller residue lysine as found in CVID instead of an arginine as found
in MVIIA. Given that relatively small differences in the sequence of
the N-type calcium channel
1B subunit can influence
-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey,
G. M., Snutch, T. P., and Zamponi, G. W. (2001)
J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID
is a N-type (Cav2.2) calcium channel variant.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxins, utilizes a four-loop framework to selectively inhibit
"N-type" voltage-sensitive calcium channels
(VSCCs)1 found in the central
and peripheral nervous systems of vertebrates (4).
-conotoxin from Conus
catus (CVID) is highly selective for N-type over P/Q-type VSCCs
(9) and shows potent analgesic activity in rats (10). In this study, we
investigated the effects of CVID on autonomic neurotransmission using
conventional intracellular microelectrode recording techniques.
-Conotoxin CVID was found to be a potent inhibitor of neurally
evoked transmitter release from the intact preganglionic cholinergic
nerves innervating the rat submandibular ganglia, whereas
-conotoxin
MVIIA had no effect.
-conotoxins
have been shown to be crucial for selective binding to the N-type VSCC
(11-15). Since the only sequence difference in loop 2 between CVID and
MVIIA is at position 10, we investigated the importance of this
position for
-conotoxin structure and ability to block
neurotransmitter release from preganglionic parasympathetic neurons.
The inhibition of preganglionic transmitter release was favored in
-conotoxins with a Lys at position 10.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
63.8 ± 0.6 mV (n = 52). The mean base line was determined by
averaging the initial part of the digitized signal between the stimulus
artifact and the onset of the response. Data are expressed as the
mean ± S.E., and n values refer to the number of
preparations. Data were analyzed statistically using Student's paired
t test with the level of significance being taken as
p < 0.05.
1B and
3 (a gift
of D. Lipscombe, Brown University) and rabbit
1C and
2
(a gift of G. Zamponi, University of Calgary) were
synthesized using a mMESSAGE mMachine in vitro transcription
kit (Ambion). Xenopus laevis stage V-VI oocytes
were removed and treated with collagenase (Sigma type I) for
defolliculation. For N- or L-type Ca2+ channel expression,
the oocytes were then injected with cRNA mixtures of either
1B (2.5 ng/cell) or
1C (5 ng/cell) in
combination with
2
and
3 in the ratio
of 1:1:1 or 1:2.5:1, respectively. For R-type Ca2+
channels, cDNA for rat
1E (4.5 ng/cell) (a gift of
G. Zamponi, University of Calgary) was injected intranuclearly in
combination with the cRNA injection of
2
and
3 (2.5 ng/cell each). The oocytes were incubated at
18 °C in ND96 solution (in mM): 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, 5 pyruvic acid, and 50 µg/ml gentamicin, pH 7.5, prior to recording. 3-8 days after
cRNA/cDNA injection, whole cell Ca2+ channel currents
were recorded from oocytes using the two-electrode (virtual ground
circuit) voltage clamp technique. Microelectrodes were filled with 3 M KCl and typically had resistances of 0.3
1.5 megohms.
All recordings were made at room temperature (20
23 °C) using bath
solution containing the following components (in mM): 5 BaCl2, 85 tetraethylammonium hydroxide, 5 KCl, and 5 HEPES titrated to pH 7.4 with methansulfonic acid. During recording,
oocytes were perfused continuously at a rate of ~1.5 ml/min. The
activation of Cl
current by Ba2+ influx
through the Ca2+ channel was eliminated by injecting 50 nl
of 50 mM BAPTA-Na4 at least 15 min prior to
recording. Using a GeneClamp 500B amplifier and pCLAMP 8 software (Axon
Instruments Inc.), data were acquired at 10 kHz after low pass
filtration at 1 kHz and leak-subtracted on-line using a P/4 protocol
and analyzed. Currents were evoked with 100-ms depolarizing pulses to 0 mV from a holding potential of
80 mV at 20-s intervals. Peak current
was measured before and during the perfusion of
-conotoxins and
antagonists for up to 10 min.
-Conotoxin Synthesis--
-Conotoxins CVID, [K10R]CVID,
MVIIA, [R10K]MVIIA, and [Y13F]CVID were assembled by manual
stepwise synthesis using in situ neutralization with Boc
chemistry (19). Starting from methylbenzhydrylamine HCl resin
(0.5-mmol scale), 4 eq of Boc-protected amino acids were coupled to the
growing chain using 4 eq of 0.5 M HBTU in N',N'-dimethylformamide and 5 eq of
N,N-diisopropylethylamine. The side chain
protection chosen was Arg(Tos), Asp(OcHex), Lys(CIZ), Thr(Bzl),
Tyr(BrZ), Ser(Bzl), and Cys(p-MeBzl). The chain assembly was monitored
after each step by the quantitative ninhydrin method and double-coupled
when coupling yields were <99.7%. The capping of the remaining amino
groups by acetylation was performed using acetic anhydride in
N',N'-dimethylformamide (87 µl/ml). After assembly, side chain-protecting groups were removed and the peptide was
concurrently cleaved from the resin using anhydrous hydrogen fluoride/p-cresol/p-thiocresol (18:1:1). The peptides were
purified and oxidized as described previously (15). Mass spectra were measured on a time-of-flight mass spectrometer (PerSeptive Biosystems) equipped with an electrospray atmospheric pressure ionization (ESI)
source. Boc-L-amino acids were obtained from Auspep
(Melbourne, Australia). Methylbenzhydrylamine HCl (0.79 mmol/g) was obtained from Peptide Institute (Osaka, Japan). HBTU was
obtained from Richelieu Biotechnologies (Quebec, Canada).
Trifluoroacetic acid, N',N'-dimethylformamide,
and N,N-diisopropylethylamine were of peptide
synthesis grade and purchased from Auspep. Acetonitrile and methanol
(Hypersolve Far-UV grade) were from BDH (Poole, United Kingdom).
Hydrogen fluoride was supplied by Boc Gases (Brisbane, Australia). All other chemicals were of analytical grade from commercial suppliers.
chemical shifts for the native peptides and the
two analogues were obtained from spectra recorded in 95%
H2O, 5% D2O, pH 3.0-3.5, at 293 K. Restraints
for three-dimensional structure calculations were obtained from spectra
recorded at 293 K with additional experiments run at 280 K to resolve
overlapping signal (15). Secondary H
chemical shifts were analyzed
compared with the random coil shift values of Wishart et al.
(20), and structures were calculated using torsion angle
dynamics/simulated annealing protocol in XPLOR version 3.8 (21-24) as
previously described (14).
20 min). The dose-response relations were determined for
-conotoxins, whereas other drugs were bath-applied at
a maximally effective concentration. In addition to the synthesized
-conotoxins, we tested
-agatoxin IVA,
-conotoxin GVIA,
-conotoxin MVIIC (Alamone Laboratories, Jerusalem, Israel), SNX-482
(Peptide Institute Inc., Osaka, Japan), cadmium chloride,
hexamethonium, mecamylamine, nifedipine, and tetrodotoxin (Sigma).
-Grammotoxin SIA was a kind gift from Dr. Rick Lampe (Zeneca
Pharmaceuticals, Wilmington, DE).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
50% of total number of neurons) (Fig.
1A) or where the EPSP does not
usually reach threshold for the initiation of an action potential (weak input synapse, ~ 25% of neurons studied) were used. EPSPs recorded from neurons receiving either strong or weak inputs were abolished by
hexamethonium (100 µM) and mecamylamine (10 µM) indicating that EPSPs were mediated by acetylcholine
acting at nicotinic receptors.
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Fig. 1.
Effect of -conotoxin
CVID on nerve-evoked EPSPs and spontaneous EPSPs in the rat
submandibular ganglion. A, control EPSPs evoked
by trains of 10 stimuli at 1 Hz. B, postsynaptic responses
evoked 20 min after the bath application of 30 nM CVID.
C, postsynaptic responses evoked 20 min after the bath
application of 300 nM CVID. Inset, effect of
CVID (100 nM) on spontaneous EPSPs. D,
dose-response relationship obtained for the effects of the calcium
channel antagonists,
-conotoxin CVID,
-conotoxin GVIA,
-conotoxin MVIIA,
-grammotoxin SIA, and
-agatoxin IVA on EPSP
peak amplitude. The solid lines represent the best fit of
the data for each toxin.
-Conotoxin
CVID produced a reversible and concentration-dependent
inhibition of EPSPs (Fig. 1). Control EPSPs from both strong and weak
input neurons had a mean amplitude of 26.2 ± 2.5 mV, and 20 min
after bath application of CVID (100 nM), these EPSPs were
reduced by 66% to a mean amplitude of 9.1 ± 1.1 mV
(n = 13). The IC50 for inhibition of the
CVID-sensitive component (79 ± 8% of peak amplitude) was 32 nM (pIC50 7.5 ± 0.15). CVID had no detectable effect on the resting membrane potential of individual ganglion cells or on the amplitude and frequency of spontaneous EPSPs
(n = 5) (Fig. 1, inset), suggesting that it
acted presynaptically to inhibit neurally evoked transmitter release.
To confirm that Tyr-13 in loop 2 contributes significantly to the
ability of CVID to inhibit transmitter release, we assessed the potency
of the phenylalanine analogue of CVID on transmitter release.
[Y13F]CVID (0.1-1 µM) was without effect on the EPSPs
(n = 3).
-conotoxin GVIA (N-type), 100 nM
-agatoxin IVA, 300 nM
-conotoxin MVIIC (P/Q-type), 300 nM
-grammotoxin SIA, 300 nM SNX-482 (R-type), and 30 µM nifedipine (L-type), which had little or no inhibitory effect on evoked and spontaneous transmitter release in rat
submandibular ganglia either alone or in combination with each other
(n = 3-13 per drug) (Fig. 1D), confirming
previous results (8). In a proportion of weak input neurons, GVIA
inhibited EPSPs by up to 12% of the control amplitude (5 of 8 preparations) (Fig. 1D) with an IC50 of 60 nM, suggesting that GVIA-sensitive calcium channels play a
minor role in neurotransmission in the rat submandibular ganglia. GVIA
had no effect on strong input neurons (n = 4). EPSPs were consistently abolished by low concentrations of the nonspecific calcium channel blocker Cd2+ (30 µM,
n = 8), indicating that neurally evoked transmitter
release from preganglionic neurons was dependent on calcium influx
through VSCCs.
-conotoxins, CVID most closely resembles MVIIA
in structure. Despite this structural similarity, MVIIA (10-1000
nM) failed to produce detectable inhibition of EPSPs (n = 4) (Fig.
2B). Since loop 2 contributes
mostly to
-conotoxin potency at N-type VSCCs, we investigated the
influence of a Lys/Arg swap at position 10 in CVID and MVIIA on their
ability to block neurotransmitter release. Bath application of a
maximally effective concentration of [K10R]CVID (100 nM)
produced only 19% inhibition of EPSPs (Fig. 2A), reducing
control EPSPs of 27.6 ± 2.9 mV to 22.4 ± 2.2 mV after 20 min (n = 8). In contrast to the ineffective MVIIA,
[R10K]MVIIA caused a 47% inhibition of EPSP amplitude (Fig. 2C), reducing control EPSPs from 27.0 ± 2.4 mV to
14.4 ± 2.2 mV after 20 min (n = 6). [R10K]MVIIA
had no detectable effect on the membrane potential of individual
ganglion neurons or on the amplitude and frequency of spontaneous EPSPs
(data not shown), suggesting that similar to CVID, it acted
presynaptically to inhibit evoked neurotransmitter release.
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Fig. 2.
Effect of [K10R]CVID,
-conotoxin MVIIA, and [R10K]MVIIA on EPSPs in the
rat submandibular ganglion. EPSPs were evoked by trains of 10 stimuli at 0.5 Hz. A, 10 superimposed traces in the absence
(control) and presence of 100 nM [K10R]CVID.
B, 10 superimposed traces in the absence
(control) and presence of 100 nM MVIIA.
C, 10 superimposed traces in the absence
(control) and presence of 100 nM [R10K]MVIIA.
D, histogram of the percentage inhibition of EPSP amplitude
by equivalent concentrations (100 nM) of CVID,
[K10R]CVID, MVIIA, and [R10K]MVIIA.
-conotoxin GVIA (100 nM),
-agatoxin
IVA (100 nM),
-conotoxin MVIIC (100 nM),
-grammotoxin SIA (100 nM), SNX-482 (100 nM),
and nifedipine (10 µM) were also investigated on the
CVID-resistant transmitter release. These toxins, applied alone or in
combination with each other, had no detectable effects on the
transmitter release recorded in the presence of 100 nM CVID
(n = 3 for each toxin).
-conotoxins
CVID, MVIIA, and their analogues for N-type VSCCs was determined from
their ability to displace [125I]GVIA from rat brain
membrane. The pIC50 values are shown in Table
I. [K10R]CVID and [R10K]MVIIA
had potencies that were comparable with the native peptides. However,
[Y13F]CVID was significantly less potent than CVID, confirming that
the hydroxyl group in Tyr-13 of CVID has a similar influence on binding
to the N-type VSCC as seen previously for the Y13F analogues of GVIA
and MVIIA (25).
Sequences and potencya of synthetic
-conotoxins
-Conotoxins on Recombinant Ca2+ Channels
Expressed in Xenopus Oocytes--
The effect of
-conotoxins CVID
and MVIIA were examined on L-, N-, and R-type Ca2+ channels
expressed in Xenopus oocytes. Whole cell Ca2+
channel currents were recorded from oocytes injected with either rat
cRNA or cDNA for
1B,
1C, or
1E in combination with
2
and
3 using 5 mM Ba2+ as the charge
carrier. Bath application of either
-conotoxins CVID or MVIIA (300 nM) inhibited the depolarization-activated inward
Ba2+ current recorded from oocytes expressing N-type but
had no effect on current through L- or R-type Ca2+ channels
(see Table II) (29-36). The inhibition
of Ba2+ currents through N-type Ca2+ channels
by
-conotoxin CVID and MVIIA was dose-dependent and slowly reversed upon washout.
Specificity of calcium channel antagonists to inhibit VSCC subtypes and
nerve-evoked transmitter release from rat preganglionic nerve
terminals
), or unknown (?)
are indicated. Numbers of replicate experiments (n) in the
present study or reference numbers are shown.
chemical shifts relative to
random coil values are a sensitive measurement of backbone conformation
for disulfide-bonded isomers and can be used to locate local
conformational differences across structurally related peptides
(25, 26). H
chemical shifts were similar for [K10R]CVID and
[R10K]MVIIA compared with the native peptides CVID and MVIIA (Fig.
3A), indicating that any
global structural changes on swapping residue 10 are minimal. The
three-dimensional structure of [R10K]MVIIA differed from MVIIA by a
root mean square deviation of 0.461 across the heavy backbone atoms,
confirming the absence of any significant structural change. This is
seen in the ribbon diagram showing the superimposition of the 17 lowest
energy conformations and the orientation of Tyr-13 and Lys/Arg-10 for
CVID, MVIIA, and [R10K]MVIIA (Fig. 3B). To determine
whether the difference in selectivity might arise from difference
surface exposure at position 10, we calculated the solvent-accessible
surface area for residue 10 over the 17-20 lowest energy conformations
in Insight II. The rank order of the solvent-accessible surface area
was MVIIA > [R10K]MVIIA > CVID (156 ± 8.0, 147 ± 8.1, and 92 ± 4.6 Å2, respectively). A complete
three-dimensional structure was not calculated for [K10R]CVID,
because it had the same potency at the N-type VSCC and conserved H
chemical shift differences compared with CVID especially across loop 2 (Fig. 3A).
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Fig. 3.
Three-dimensional structures of
-conotoxins CVID, MVIIA, and [R10K]MVIIA.
A, H
chemical shifts for
-conotoxins CVID,
[K10R]CVID, MVIIA, and [R10K]MVIIA at 293 K in 95%
H2O, 5% D2O. Residue numbers are labeled
according to the CVID sequence. B, ribbon diagrams of the
backbones for CVID (i), MVIIA (ii), and
[R10K]MVIIA (iii). The 17 lowest energy conformations of
the side chains at positions 10 (Lys or Arg) and 13 (Tyr) are also
shown on the left side. On the right side are
shown the ribbon diagrams rotated 180o around the
y axis and the calculated Connolly surface area of
Lys/Arg-10 and the orientation of Tyr-13 in loop 2 (residues 8-14 are
shown). Structures of the lowest energy conformation were superimposed
over backbone atoms C
, C, and N (1-27 and 1-25) for CVID, MVIIA,
and [R10K]MVIIA, respectively (Insight II 2000).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxin CVID,
the most selective N-type versus P/Q-type VSCC blocker, on
transmitter release from these nerve terminals. CVID (IC50 = 32 nM) produced up to 80% inhibition of nerve-evoked
transmitter release without affecting spontaneous transmitter release,
and this inhibition was reversed upon washout. In contrast,
nerve-evoked transmitter release was unaffected by MVIIA (
1
µM). Interestingly, the potency of CVID to inhibit
transmitter release from preganglionic cholinergic neurons was similar
to the potency of both CVID and MVIIA to inhibit N-type VSCCs in
postganglionic sympathetic neurons of rat vas deferens (9).
-conotoxins has been identified as the primary loop
involved in binding to the N-type VSCC (11, 13, 25) with the hydroxyl
group on Tyr-13 on both
-conotoxin GVIA and MVIIA found to be a
critical determinant of high interaction at the N-type VSCC (12, 27).
The 400-fold reduction in potency of [Y13F]CVID for rat brain N-type
VSCC confirmed a key role of Tyr-13 in CVID. [Y13F]CVID was also
inactive on the EPSPs, indicating that N-type and CVID-sensitive VSCCs
both require the hydroxyl of Tyr-13. One possible conclusion from this
result is that CVID may bind to both VSCCs in a similar manner. Because
loop 2 of MVIIA and CVID vary only at position 10 (Lys or Arg,
respectively) (see Table. 1), we investigated the role of this position
contributing to the selectivity differences observed between MVIIA and
CVID by swapping these residues. [K10R]CVID (100 nM)
produced significantly less (< 20%) inhibition of transmitter release
than CVID, indicating that Lys-10 was the preferred residue for
resistant-type VSCC inhibition by CVID. Supporting this conclusion,
Lys-10 was found to markedly enhance MVIIA inhibition of resistant-type
VSCCs with the [R10K]MVIIA (100 nM) causing 47%
inhibition of evoked transmitter release (i.e. >100-fold
more than MVIIA). None of the
-conotoxins investigated affected
spontaneous transmitter release or the resting membrane potential of
the postsynaptic cell, indicating a selective action at presynaptic VSCCs.
-conotoxin GVIA,
-conotoxin MVIIC,
-agatoxin IVA,
-grammotoxin SIA, and SNX-482) were investigated on the transmitter
release remaining after the application of CVID. None of these VSCC
inhibitors, either applied alone or in combination, had any effect on
the CVID-resistant neurotransmitter release. However, the
CVID-resistant release was reversibly abolished by Cd2+,
indicating its dependence on calcium entry into nerve terminals through
VSCCs. Because the R-type VSCC inhibitors used do not inhibit all
R-type VSCCs (28), we investigated the effects of
-conotoxins CVID
and MVIIA on current flow-through cloned L- or R-type calcium
channels expressed in Xenopus oocytes. Both CVID and
MVIIA had no effect on depolarization-activated Ba2+
currents through L- or R-type calcium channels but potently inhibited current through N-type VSCCs (see Table II) as reported
previously (9).
-conotoxin MVIIA and [R10K]MVIIA at the
CVID-sensitive VSCCs, we determined their secondary and tertiary
structures by 1H NMR. From these studies, it was evident
that residue replacements at position 10 had little effect on the
global fold or structure of CVID or MVIIA. However, examining the
solvent-accessible surface area at position 10 revealed a trend of
decreasing surface area that correlated with activity at the
CVID-sensitive VSCCs in preganglionic neurons. It is possible that the
degree of exposure may influence the selectivity of
-conotoxins with
the most exposed residue, Arg in MVIIA, creating a clash that precludes
binding to certain VSCCs. Recently, Feng et al. (37)
identified that a single residue change on an extracellular loop of the
N-type VSCC (G1326) can influence
-conotoxin access. Thus,
relatively small differences in N-type VSCC sequence may significantly
alter
-conotoxin affinity, raising to the possibility that the VSCC
targeted by CVID in preganglionic nerve terminals may be a N-type
(Cav2.2) calcium channel variant that does not bind all
-conotoxins. Two functionally distinct variants of the
1B subunit,
1B-b and
1B-d,
of the N-type calcium channel that differ at two loci (four amino acids
(SFMG) in IIIS3-S4 and two amino acids (Glu/Thr) in IVS3-S4)
have been shown to be reciprocally expressed in rat brain and
sympathetic ganglia (38). The selective expression of pharmacologically
distinct VSCC splice variants of
1B subunits in
different regions of the nervous system is proposed to underlie the
differential block of preganglionic nerve terminal VSCCs by N-type
selective
-conotoxins. Alternative splicing of the domain II-III
linker region of the human N-type (Cav2.2) calcium channel
1B subunit has recently been shown to differentially
affect
-conotoxin MVIIA and GVIA block of the calcium channel
current (39).
-conotoxin affinity for
this N-type-related VSCC. Given that [125I]CVID labels
fewer sites in rat brain than [125I]MVIIA (9), it is
unlikely that these resistant-type VSCCs are among the high
voltage-activated VSCCs labeled by [125I]
-Aga-IIIA in
rat brain (40). Intrathecal CVID (AM336) and MVIIA (Ziconotide) are
currently being evaluated clinically as treatments for severe pain (10,
41). Because parasympathetic nerves arise from spinal cord neurons, it
is possible that CVID-sensitive VSCCs are also expressed and play a
role in neurotransmission in the spinal cord. Given the differences in
the selectivity of
-conotoxin CVID and MVIIA for this VSCC subtype,
these
-conotoxins may have different profiles of activity in the
central (for review see Refs. 10 and 42) as well as the peripheral
nervous system.
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FOOTNOTES |
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* This work was supported in part by the Australian Research Council and National Health and Medical Research Council of Australia.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.
§ To whom correspondence should be addressed: School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. Tel.: 61-7-3365-1074; Fax: 61-7-3365-4933; E-mail: dadams@mailbox.uq.edu.au.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209969200
1 The abbreviations used are: VSCC, voltage-sensitive calcium channel; EPSP, excitatory postsynaptic potential; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; BAPTA, 1,2-bis(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetate.
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