From the Departments of Physiology & Biophysics and
Pharmacology & Therapeutics, Neuroscience Research Group, University of
Calgary, Calgary, Alberta T2N 4N1, Canada, § NeuroMed
Technologies Inc., Vancouver V6T 1Z4, Canada, and the ** Biotechnology
Laboratory, University of British Columbia, Vancouver
V6T 1Z3, Canada
Received for publication, January 16, 2001, and in revised form, February 2, 2001
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ABSTRACT |
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We recently reported that amino acid residues
contained within a putative EF hand motif in the domain III S5-H5
region of the It is well established that peptide toxins isolated from marine
snails have the propensity to potently and highly selectively inhibit
the activities of a number of different types of voltage- and
ligand-gated ion channels (1-5). These toxins include blockers of
skeletal muscle sodium channels (µ-conotoxin (6-8)), the nicotinic acetylcholine receptor ( While little is presently known concerning the toxin structural
requirements, which underlie the high affinity and selectivity of
We have recently reported that point mutations in an external EF hand
homology motif located on the N-type calcium channel Site-directed Mutagenesis--
The G1326P, E1332R, D1323R, and
E1321R point mutation constructs have been described by Feng et
al. (18). The R1325D, G1326K, G1326E, and G1326Y additional
constructs were generated as described by Feng et al. (18).
In brief, a SplI-AflII fragment (~4.5
kilobases) was excised from the full-length
cytomegalovirus- Tissue Culture and Transient Transfection--
Human embryonic
kidney tsa-201 cells were maintained in a 37 °C
CO2 incubator in standard Dulbecco's modified Eagle's
medium (supplemented with 10% fetal bovine serum, 200 units/ml
penicillin, and 0.2 mg/ml streptomycin). The cells were split at 85%
confluence using trypsin EDTA and plated on glass coverslips at 5-10%
confluence. The cells were then allowed to recover for ~12 h at
37 °C before transfection. Immediately before transfection, the
medium was replaced, and the cells were transiently transfected with
cDNAs encoding for calcium channel Whole Cell Patch Clamp Recordings and Data Analysis--
The
whole cell patch clamp recording (membrane ruptured) procedures used
here were similar to those described by Beedle and Zamponi (19).
Briefly, cells were transferred to a 3-cm culture dish containing
recording solution comprised of 20 mM BaCl2 (or CaCl2), 1 mM MgCl2, 10 mM HEPES, 40 mM
TEA1-Cl, 10 mM
glucose, 87.5 mM CsCl (pH 7.2 with TEA-OH). Pipettes with
resistances of 2-4 M Mutations in the Domain III S5-H5 Region Affect
In our previous study examining permeation, we replaced the central
glycine residue (Gly1326) within the putative external EF
hand domain with proline (18). To determine whether this substitution
affects block of the channel by
To determine whether this effect is also observed with other
substitutions at the G1326 position, we generated three additional mutations (G1326K, G1326E, and G1326Y). Interestingly, the G1326Y mutant resulted in channels that could no longer be functionally expressed. However, the G1326K and G1326E mutants expressed well, and
as shown in Fig. 5A and B, the time
course of development of block was accelerated, albeit not as
dramatically as with the G1326P mutant. In both cases, block remained
irreversible over the time course of a typical experiment. This is also
evident from the kinetic analysis presented in Fig. 5, C and
E, where the slope of the regression line (and hence, the
blocking rate constant) is clearly increased, whereas the intercept on
the ordinate is similar to that obtained with the wild type
channels. To test whether the presence of a proline residue per
se was sufficient to render the channels reversible from
Overall, our data indicate that at least two additional residues
besides those identified previously by Ellinor et al. (16) are important determinants of The G1326P Mutant Results in Reversible Peptide toxins isolated from venomous animal species are
among the most potent inhibitors of voltage-gated calcium channels (2,
21). Calcium channel blocking peptides appear to fall into two major
classes: gating blockers, which allosterically inhibit channel opening
(e.g. We have previously reported that the expression of the G1326P, E1332R,
and a double (E1321K,D1323R) mutant resulted in currents that no longer
supported a differential permeability to calcium and barium ions, but
otherwise displayed biophysical properties that did not differ
significantly from those of the wild type channels, including
activation, inactivation, and block by divalent cations (18). We
attributed the effects on ion selectivity to a highly localized
specific disruption of the function of a putative calcium binding EF
hand spanned by residues 1321-1332, rather than a global structural
rearrangement of the The most pronounced effect on toxin action was observed with the G1326P
construct in that the blocking rate constant was increased 16-fold, and
block became entirely reversible. This suggests that the mutation
increases both the accessibility of the binding site and at the same
time results in a less stable interaction between the toxin and the
channel. Interestingly, other substitutions in this position, while
increasing the rate of development of block did not affect the
reversibility of block. It is well established that proline residues
act as helix breakers and thus may mediate a more pronounced effect on
the local structure of the channel. However, the presence of a proline
in this region of the channel per se is not sufficient to
render toxin block reversible, since the E1332P construct did not
affect recovery from toxin block. The dramatic effects on the
reversibility of block seen with the G1326P mutant cannot be explained
by a simple change in toxin affinity, since this mutation resulted in
only a ~4-fold increase in the equilibrium dissociation constants of
the two toxins as determined from the ratios of unblocking and blocking
time constants. Instead, we envision a scenario in which residue
Gly1326 restricts the access of the toxin to its docking
site (Fig. 7). Once the toxin has docked
to its receptor site, the barrier formed by Gly1326 would
aid in the stabilization of the channel toxin interaction. Upon
mutation of Gly1326 to proline, this barrier may be
removed, thereby providing greater access to the blocking site, but
concomitantly resulting in a less stable interaction. Substitutions of
Gly1326 with other types of amino acid residues may not be
as effective in removing the putative access barrier and, hence, affect
the time constant for development of block and unblock to a smaller degree.
1B subunit affected the relative
barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M.,
Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001)
J. Biol. Chem. 276, 5726-5730). Since this region
partially overlaps with residues previously implicated in block of the
channel by
-conotoxin GVIA, we assessed the effects of mutations in
the putative EF hand domain on channel block by
-conotoxin GVIA and
the structurally related
-conotoxin MVIIA. Both of the toxins
irreversibly block the activity of wild type
1B N-type
channels. We find that in addition to previously identified amino acid
residues, residues in positions 1326 and 1332 are important determinants of
-conotoxin GVIA blockade. Substitution of residue Glu1332 to arginine slows the time course of
development of block. Point mutations in position Gly1326
to either arginine, glutamic acid, or proline dramatically decrease the
time constant for development of the block. Additionally, in the
G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with
-conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly1326 may form a barrier, which controls the
access of peptide toxins to their blocking site within the outer
vestibule of the channel pore and also stabilizes the toxin-channel interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxins (9, 10)), and blockers of
voltage-dependent calcium channels (
-conotoxins (2)).
Among the family of
-conotoxins, two prominent members are
-conotoxin GVIA, a 27-amino acid peptide derived from the
Conus geographus sea snail, and
-conotoxin MVIIA, a
25-amino acid toxin isolated from the venom of the Conus
magus marine snail (2). Both of these toxins potently block N-type
calcium channels from a variety of species and nerve tissues (11-14).
Block develops rapidly and is only incompletely reversible even after
prolonged washout (12). Both toxins have been reported to display a
high degree of selectivity for the N-type calcium channel
1B subunit (15), and
-conotoxin GVIA has become a
commonly used tool for the identification of native N-type calcium currents.
-conotoxins GVIA and MVIIA for N-type calcium channels, some of the
N-type calcium channel regions that participate in the blocking
interaction have been elucidated. Utilizing a chimeric channel approach
in combination with site-directed mutagenesis, Ellinor and co-workers
(16) identified crucial components of the binding site of
-conotoxin
GIVA in the external vestibule of the
1B channel in the
domain III S5-S6 region, suggesting that this toxin likely acts via
physical occlusion of the pore. While the binding site for
-conotoxin MVIIA has not yet been identified via molecular
biological techniques, both biochemical and electrophysiological data
suggest that
-conotoxin MVIIA competes with
-conotoxin GVIA
for a common, or at least partially overlapping, receptor site
(2, 17).
1B
subunit immediately adjacent to the
-conotoxin GVIA binding site
identified by Ellinor et al. (16) affects the permeation characteristics of the channel (18). In view of its proximity to the
putative
-conotoxin GVIA receptor site, we investigated whether
mutations in this region could affect the characteristics of
-conotoxin GVIA and MVIIA block of the channel. We report here that
certain point mutations within the putative EF hand motif increase the
affinity of the channel for
-conotoxin GVIA. Moreover, one of the
mutations, a replacement of residue Gly1326 with proline,
is sufficient to dramatically enhance the unblocking rate of the toxin
such that block by both
-conotoxin GVIA and MVIIA is fully
reversible over a time course of <10 min. Hence, residues immediately
outside the region identified previously by Ellinor et al.
(16) contribute to block of N-type calcium channels by
-conotoxins,
and a single amino acid residue in this region is sufficient to
control the reversibility of toxin block.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1B (CMV) construct and ligated
into pSL1180. Site-directed mutagenesis was carried out on using the
QuikChange mutagenesis kit (Stratagene) and successful mutagenesis,
and absence of sequence errors was confirmed via DNA sequencing.
Subsequently, the mutated SplI-AflII fragment was
introduced into the full-length clone in the cytomegalovirus (CMV) expression vector via restriction enzyme digestion an ligation and its presence confirmed by restriction endonuclease digestion and
DNA sequencing.
1B,
1b, and
2-
subunits and EGFP (at a molar ratio of 1:1:1:0.3) via the calcium phosphate method. After
12 h, the cells were washed with fresh medium, allowed to recover
for additional 12 h, and subsequently stored at 28 °C in 5%
CO2 for 1-3 days before whole cell recording. Recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) linked to a personal computer equipped with pClamp version 7.0. Patch pipettes (Sutter borosilicate glass, BF 150-86-15) were pulled using a Sutter P-87 microelectrode puller and subsequently fire-polished using a Narashige microforge.
were filled with internal recording solution
containing 108 mM cesium methanesulfonate, 4 mM
MgCl2, 9 mM EGTA, 9 mM HEPES (pH
7.2 with TEA-OH). After patch rupture, cell capacitance was
neutralized, and series resistance was compensated. Currents were
elicited by stepping from a holding potential of
100 mV to various
test potentials using Clampex 7.0 software and an Axopatch 200 B
amplifier (Axon Instruments, Foster City, CA).
-Conotoxin GVIA (RBI
Chemicals) and MVIIA (Sigma) were dissolved first, respectively, in
water and acetic acid and then diluted in the external recording
solution. Solutions containing various concentrations of toxin were
delivered directly to the cells by means of a gravity-driven
microperfusion system, which allows complete solution exchanges in less
than 1 s. Currents underwent little if any rundown during the time
course of a typical experiment. Data were filtered at 1 kHz and
digitized at a sampling frequency of 2 kHz. Data were analyzed using
Clampfit (Axon Instruments). All curve fittings were carried out using
Sigmaplot 4.0 (Jandel Scientific). The time constant,
, for onset of
block was determined from the equation
I(t)/I(t = 0) = exp(
t/
), where I(t) is the current amplitude at time t after application of the toxin,
and I(t = 0) is the control current
amplitude immediately prior to toxin application. Statistical analysis
was carried out using SigmaStat 2.0 (Jandel Scientific). Differences
between mean values from each experimental group were tested using a
Student's t test for two groups and one way analysis of
variance for multiple comparisons. Differences were considered
significant if p < 0.05. All error bars given reflect
S.E values, numbers in parentheses indicate numbers of cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Conotoxins GVIA and MVIIA Irreversibly Block N-type Calcium
Channels--
Fig. 1 illustrates the
effects of
-conotoxin GVIA and MVIIA on N-type
(
1B+
1b+
2-
) calcium
channels transiently expressed in HEK tsa-201 cells. The application of
1 µM of either toxin completely removes current activity,
which cannot be recovered following washout over the time course of 10 min. Fig. 1B compares the time courses of N-type channel
block for various concentrations of both toxin. For both toxins the
time course of development of block is accelerated with increasing
toxin concentration, although on average development of
-conotoxin
MVIIA block occurs about three times more rapidly than that of
-conotoxin GVIA. Similar to what has been previously reported for
both native channels (14) and
1B channels transiently
expressed in Xenopus oocytes (16), block of both toxins was
only poorly reversible such that even prolonged washout (7-15 min)
resulted in the recovery of only a minor fraction of the N-type
current. Fig. 1C illustrates the dependence of the inverse
of the time constant for development of block on the toxin
concentration. Consistent with a bimolecular interaction between the
toxin molecule and the channel, the relation was well described by a
linear fit in the case of each of the two toxins. The blocking rate
constants (kon), obtained from the regression
lines, were, respectively, 1.06 and 2.93 µM
1
min
1 for
-conotoxins GVIA/MVIIA,
consistent with the data shown in Fig. 1B. However, it is
not possible to accurately determine the unblocking rate constants
(koff) from either the regression lines or from
the time course of recovery from block because of the poor
reversibility of the blocking action.
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Fig. 1.
Block of transiently expressed N-type
( 1B+
1b+
2-
)
calcium channels by
-conotoxins
(CTX) GVIA and MVIIA. A, current
records illustrating the effects of the two toxins. Block is complete
and virtually irreversible in response to washout for 10 min. Currents
were elicited by stepping from a holding potential of
100 mV to a
test potential of +20 mV. B, representative time courses of
development of block and of recovery from block by
-conotoxin GVIA
and
-conotoxin MVIIA. In both cases, the block develops more rapidly
at higher toxin concentrations. Note that block by
-conotoxin MVIIA
develops more rapidly than that of
-conotoxin GVIA. C,
dependence of the inverse of the time constant for development of
block,
, on the toxin concentration, [CTX]. The
solid lines are linear regressions (fitted with the
equation: 1/
= kon*[conotoxin] + koff), whose slopes reflect the blocking rate
constants (kon) and whose intercepts on the
y axis are equivalent to the unblocking rate constants
(koff). The rate constants obtained from the
fits are as follows:
-conotoxin GVIA, kon = 1.06 µM
1
min
1, koff = 0.011 min
1;
-conotoxin MVIIA,
kon = 2.93 µM
1
min
1, koff = 0.07 min
1). Error bars denote S.E.
values.
-Conotoxin GVIA
Block--
We have previously reported that high voltage-activated
calcium channels contain a highly conserved putative EF hand motif within the domain III S5-H5 region (18). As shown in Fig.
2 for the
1B subunit, this
region is localized immediately adjacent to a series of amino acid
residues, which have been implicated in
-conotoxin GVIA block of the
channel (16). In a previous study we showed that replacement of the
three negative charges in the putative EF hand motif with positively
charged residues, or substitution of the central glycine
(Gly1326), resulted in an ablation of the differential
selectivity of the channel between barium and calcium ions without
affecting other major biophysical properties of the channel such as
activation, inactivation, or block by cadmium ions (18). To test
whether this region of the channel might modulate the blocking action of
-conotoxin GVIA, we first expressed the triple
(E1321K,D1323R,E1332R)
1B mutant in HEK tsa-201 cells
(together with the ancillary
1b and
2-
subunits) and examined the dose dependence of
-conotoxin GVIA
blocking kinetics of the mutant channel. As shown in Fig. 3, the time course of development of
-conotoxin GVIA block in the triple mutant channel was significantly
slowed compared with that observed with the wild type channel,
indicating that one or a combination of the three substituted residues
contribute to
-conotoxin GVIA block of the channel. Nonetheless, the
application of 1 µM concentrations of the toxin was
sufficient to eliminate all current activity, and as with the wild type
channel, block of the mutant channel was irreversible (Fig. 3,
A and B). To determine which of the three
residues was responsible for mediating the slowing of the blocking
kinetics, we examined
-conotoxin GVIA block of both a single E1332R
and a double (E1321K,D1323R) mutant. As seen in Fig. 3B, a
single substitution of residue 1332 with arginine was sufficient to
account for the slowing of the blocking kinetics, whereas the double
mutant behaved similar to the wild type channel. This is shown
quantitatively in form of a comparison of the blocking rate constants
of
-conotoxin GVIA block of the wild type and the mutant channels
(Fig. 3C). Mutation in position Glu1332 to
arginine resulted in a 2-fold reduction in kon
and suggests that the glutamate contributes in a significant manner to
-conotoxin GVIA block of N-type calcium channels.
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Fig. 2.
A, amino acid sequence of the
1B domain III S5-H5 region. The putative EF hand calcium
binding domain (
) spans residues Glu1321 to
Glu1332. Amino acid residues implicated previously in
-conotoxin (CTX) GVIA block (16) (
) are located
between Gln1327 and Gln1339. B,
proposed membrane topology of the N-type calcium channel
1 subunit (20) indicating the location of the putative
EF hand motif. The inset illustrates the possible locations
of the key residues relative to the pore forming p-loop structure of
the channel as proposed by Ellinor et al. (16).
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Fig. 3.
Effect of mutations in positions 1321, 1323, and 1332 on toxin block. A, current records obtained
with mutant (E1321K,D1323R, E1332R), (E1321K,D1323R), or E1332R
channels (coexpressed with 1b and
2-
)
in the absence and presence of 1 µM
-conotoxin GVIA.
Note that all three mutants are irreversibly and completely blocked at
this toxin concentration. B, representative time course of
development of block of wild type and mutant N-type calcium channels by
1 µM
-conotoxin GVIA. Note that the triple mutant and
the single E1332R mutant channel display slowed blocking kinetics
compared with the wild type or the (E1321K,D1323R) mutant construct.
C, blocking rate constants obtained with the wild type and
mutant channels. The kon values were obtained by
fitting linear regressions as shown in Fig. 1C for each
individual experiment. Error bars denote S.E. values,
numbers in parentheses reflect numbers of
experiments, and the asterisks indicate statistical
significance relative to the wild type channels (p < 0.05).
-conotoxin GVIA, the G1326P mutant
was expressed with the appropriate ancillary subunits in tsa-210 cells,
and
-conotoxin GVIA blocking kinetics were examined. Fig.
4 shows that development of block was
accelerated 30-fold compared with the wild type channel (Fig.
4B). More strikingly, upon washout of the toxin current activity could be almost completely recovered over the time course of
about 10 min (Fig. 4, A and B). Since the time
course for development of block is inversely proportional to the sum of
blocking and unblocking rates, such an increase in the rate of recovery
from block is predicted to contribute to the observed 30-fold decrease in the time constant for development of block. To isolate any direct
effects of the mutation on the true blocking rate constant, the time
course for development of block was determined at several toxin
concentrations, and the inverse of the time constant of development of
block was plotted as a function of toxin concentration. As shown in
Fig. 4C, the relation was nicely described by a linear fit.
Furthermore, from the slope and the y intercept of the
regression line, both the blocking (kon = 17.51 µM
1
min
1) and the unblocking
(koff = 0.72 min
1)
rate constants were found to be significantly increased in the G1326P
mutant compared with the wild type channel, indicating that the access
of the toxin to its binding site is enhanced in the mutant, but
concomitantly the stability of the binding interaction is dramatically
reduced.
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Fig. 4.
-Conotoxin
(CTX) GVIA block of the G1326P mutant.
A, current records illustrating block of the G1326P
(+
1b+
2-
) construct by 100 nM
-conotoxin GVIA. Note that the effects of the toxin
are almost completely reversed following washout. B,
representative time course of development of, and recovery from, block
of the G1326P mutant by different concentrations of
-conotoxin GVIA.
Note the reversibility of toxin block. C, kinetic analysis
of block of wild type and G1326P mutant channels by
-conotoxin GVIA.
The data for the wild type channel are the same as in Fig. 1. Note the
dramatic increase in the slope of the regression line and the larger
y intercept obtained with the mutant channel. The blocking
and unblocking rate constants for the G1326P mutant obtained from the
fit were, respectively, 17.5 µM
1
min
1 and 0.72 min
1.
Error bars denote S.E. values.
-conotoxin GVIA block, we generated an E1332P mutant. As shown in
Fig. 5D,
-conotoxin GVIA block of this mutant was not
reversible, hence the presence of a proline per se near in
the putative EF hand motif is insufficient to render the channels
reversible from toxin block. Interestingly, the effect of the E1332P
mutation on the blocking rate constant was opposite to that obtained
with the E1332R construct, which tended to slow the blocking
kinetics.
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Fig. 5.
A and B, effects of different
substitutions for Gly1326 on -conotoxin (CTX)
GVIA block in the form of representative time courses. Note that unlike
in case of G1326P, block of G1326K and G1326E channels remains
irreversible. C, kinetic analysis of toxin block as
described in Fig. 1C. Note that the blocking rate constant
is increased in both mutants compared with the wild type channels. The
blocking and unblocking rate constants obtained from the fits were as
follows: G1326K, kon = 2.18 µM
1
min
1, koff = 0.5 min
1; G1326E, kon = 3.06 µM
1
min
1, koff = 0.21 min
1). D, effect of a substitution
of Glu1332 to proline. Unlike the G1326P mutant, the E1332P
mutant cannot be recovered from toxin block. E, comparison
of the blocking rate constants obtained with wild type and mutant
channels. The blocking rate constants were obtained as described in the
legend to Fig. 3C. Error bars denote S.E. values,
numbers in parentheses are numbers of
experiments, and asterisks denote statistical significance
relative to control. All mutants shown in the figure were coexpressed
with
1b and
2-
subunits.
-conotoxin GVIA block and that residue
1326 is an important factor in controlling both access of the toxin and
the strength of interaction with the channel.
-Conotoxin MVIIA
Binding--
The striking effects observed with the G1326P mutant
raise an obvious question: is
-conotoxin MVIIA block affected in a
similar manner? As shown in Fig. 6, both
the development and the reversibility of block of the channel by
-conotoxin MVIIA are dramatically enhanced in the
Gly1326 mutant. However, there were subtle differences in
the manner by which the mutation affected block by the two toxins.
Whereas the effects of the mutation on recovery from
-conotoxin
MVIIA block was even more pronounced than those observed with the GVIA isoform (i.e. complete recovery from block after 5 min), the
effect of the mutation on the blocking rate constant was greater for
-conotoxin GVIA (16-fold increase in kon)
than for
-conotoxin MVIIA (7-fold increase). Nonetheless, these data
indicate that there is significant overlap in the channel structural
determinants of the access of
-conotoxins GVIA and MVIIA to their
binding sites, consistent with biochemical data showing that both
toxins compete for a common binding site (11).
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Fig. 6.
Effect of mutations in positions 1326 and
1332 on -conotoxin (CTX)
MVIIA block. A, representative time course of
development of block and recovery from block for the G1326P mutant.
Note the rapid reversibility of blocking action. B, kinetic
analysis of
-conotoxin MVIIA block of wild type
1B or
mutant (E1321K,D1323R,E1332R), and G1326P mutant channels (coexpressed
with
1b and
2-
). The blocking and
unblocking rate constants obtained from the fits were as follows:
G1326P, kon = 19.87 µM
1
min
1, koff = 2.11 min
1; triple mutant,
kon = 0.68 µM
1
min
1, koff = 0.23 min
1). C and D, blocking and
unblocking rate constants obtained with wild type, G1326P and
(E1321K,D1323R,E1332R) channels. Data were obtained via linear
regressions as shown in Fig. 1C for each individual
experiment. The error bars denote S.E. valuess,
numbers in parentheses reflect the numbers of
individual experiments, and asterisks denote significance
relative to wild type channels.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-agatoxin IVA (22, 23) and
-grammotoxin-S1A
(24-26)), and pore blockers, which physically prevent ion permeation
(e.g.
-conotoxins GVIA (16) and MVIIC (11, 17) and
-agatoxin III (27, 28)). To date, the exact sites of action of the
gating inhibitors on the calcium channel
1 subunit
remain enigmatic. Furthermore, unlike in the case of potassium channels
(5, 29) and sodium channels (7, 30-32), the detailed molecular
mechanisms by which calcium channels are inhibited by pore-blocking
toxins remain poorly understood. To date, there has only been one
comprehensive study that has examined the channel structural
determinants of
-conotoxin GVIA block. Using a series of chimeras
between
1A P/Q-type and
1B N-type calcium
channels, Ellinor et al. (16) were able to show that the
domain III S5-S6 region was critically important for block of N-type
calcium channels by
-conotoxin GVIA. In particular, residues
Gln1327, Glu1334, Glu1337, and
Gln1339 were found to contribute to
-conotoxin GVIA
block, with Glu1337 mediating the largest effect. In the
present study, we have identified two additional residues that appear
to be important for the interaction of the channel with pore-blocking
toxins, residues Gly1326 and Glu1332. Since
these residues are conserved in
1A and
1B
channels (33), their contributions to toxin block could not have been
detected with a chimeric approach. It is interesting to note that amino acid substitutions adjacent to the region identified by Ellinor et al. (16) (i.e. Glu1332 and
Gly1326) mediated a more pronounced effect on
-conotoxin
GVIA block, compared with mutations that occur further upstream
(i.e. Glu1321 and Asp1332).
This could perhaps suggest that residue 1326 might form an outer
boundary for the
-conotoxin GVIA receptor site. This is further
supported by data obtained with an R1325E construct, which did not
display altered toxin block (not shown).
1B subunit. The observation that
substitutions in positions 1321 and 1323 did not affect
-conotoxin
GVIA block is consistent with a highly localized and specific effect of
amino acid substitutions in these positions on ion selectivity.
Moreover, that substitutions in positions Gly1326 and
Glu1332 did not affect channel gating, and cadmium block
(18) is also consistent with a localized effect of these mutations on
the toxin-channel interaction.
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Fig. 7.
Possible model accounting for the effects of
the G1326P mutation on the time course of development and recovery
from -conotoxin (CTX)
block. In the wild type channel, residue Gly1326 may
form a barrier that restricts access of the toxin to its blocking site
but also prevents the dissociation of the toxin once docked to the
receptor. The introduction of the proline residue may remove this
barrier, thus providing more ready access and concomitantly, a less
stable binding interaction.
It is more difficult to speculate about the basis for the effects of
substitutions in position Glu1332. It is possible that this
residue forms a low affinity contact point between the toxin and the
channel. Alternatively, changes in this residue may allosterically
affect the interaction of the toxin with residue Glu1334,
which was previously identified as an important determinant of
-conotoxin GVIA block (16). The change in the time constant of
development of block observed with the E1332R and E1332P constructs are
unlikely due to a change in net charge of the channel, since neither
the double (E1321K,D1323R) nor the R1325E substitution significantly
affected toxin block.
A major finding is that mutations that affect block of the channel by
-conotoxin GVIA also affect the action of
-conotoxin MVIIA. To
date, the only evidence suggesting an overlap between the blocking
sites for the two toxins had come from competitive binding studies (2).
Here, we have demonstrated for the first time at the amino acid level
that the two toxins share some of the same channel molecular
determinants. The two toxins share a similar disulfide bond arrangement
comprised of six conserved cysteine residues, but only about 27% in
sequence identity among their non-cysteine residues (2, 34). This
suggests that one or more of the conserved toxin residues may perhaps
interact with residue 1326 on the calcium channels or that the
three-dimensional folding of the toxin backbone is a key determinant
for blocking action. The latter scenario would fit with the model shown
in Fig. 7, which predicts that toxins with an overall similarity in
their backbone structure would be similarly affected by the presence/absence of an access barrier. Regardless of the underlying mechanism, our data show that a single amino acid residue in the domain
III S5-H5 region is sufficient to control access of two structurally
distinct peptides from two different cone snail species, suggesting
that the basic mechanism by which these two toxins block channel
activity is highly conserved.
In summary, our data identify two residues (Gly1326 and
Glu1332) in the 1B subunit, which are
important determinants of
-conotoxin block. The G1326P mutant may
serve as a convenient tool for further investigation into the detailed
mechanisms underlying
-conotoxin block of N-type calcium channels by
allowing direct access to the effects of any additional mutations on
the channel or the toxin molecule on both blocking and unblocking
kinetics. In view of the clinical use of
-conotoxin MVIIA as an
analgesic (35, 36), such information may aid in the design of more
effective calcium channel therapeutics.
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FOOTNOTES |
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* This work was supported by operating grants (to G. W. Z.) from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Alberta and the Northwest Territories and through a Scholarship Award (to G. W. Z.) from the EJLB Foundation.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.
¶ These authors contributed equally to this study.
Recipient of a postdoctoral fellowship from the Natural
Science and Engineering Research Council of Canada.
Holds CIHR operating funds and a Senior Scientist award from
the CIHR.
§§ Holds Faculty Scholarships from the Alberta Heritage Foundation for Medical Research (AHFMR) and the CIHR and is the Novartis Investigator in Schizophrenia Research. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8687; Fax: 403-210-8106; E-mail: zamponi@ucalgary.ca.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M100406200
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ABBREVIATIONS |
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The abbreviation used is: TEA, tetraethylammonium.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Quik, M. (1992) Mol. Neurobiol. 6, 19-40[Medline] [Order article via Infotrieve] |
2. | Olivera, B. M., Miljanich, G. P., Ramachandran, J., and Adams, M. E. (1994) Annu. Rev. Biochem. 63, 823-867[CrossRef][Medline] [Order article via Infotrieve] |
3. | Favreau, P., Le Gall, F., Benoit, E., and Molgo, J. (1999) Acta Physiol. Pharmacol. Ther. Latinoam. 49, 257-267[Medline] [Order article via Infotrieve] |
4. |
Possani, L. D.,
Becerril, B.,
Delepierre, M.,
and Tytgat, J.
(1999)
Eur. J. Biochem.
264,
287-300 |
5. | Tenenholz, T. C., Klenk, K. C., Matteson, D. R., Blaustein, M. P., and Weber, D. J. (2000) Rev. Physiol. Biochem. Pharmacol. 140, 135-185[Medline] [Order article via Infotrieve] |
6. | Moczydlowski, E., Olivera, B. M., Gray, W. R., and Strichartz, G. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5321-5325[Abstract] |
7. | Becker, S., Prusak-Sochaczewski, E., Zamponi, G., Beck-Sickinger, A. G., Gordon, R. D., and French, R. J. (1992) Biochemistry 31, 8229-8238[Medline] [Order article via Infotrieve] |
8. | French, R. J., Prusak-Sochaczewski, E., Zamponi, G. W., Becker, S., Kularatna, A. S., and Horn, R. (1996) Neuron 16, 407-413[Medline] [Order article via Infotrieve] |
9. |
Kulak, J. M.,
Nguyen, T. A.,
Olivera, B. M.,
and McIntosh, J. M.
(1997)
J. Neurosci.
17,
5263-5270 |
10. |
Luo, S.,
Kulak, J. M.,
Cartier, G. E.,
Jacobsen, R. B.,
Yoshikami, D.,
Olivera, B. M.,
and McIntosh, J. M.
(1998)
J. Neurosci.
18,
8571-8579 |
11. | Woppmann, A., Ramachandran, J., and Miljanich, G. P. (1994) Mol. Cell. Neurosci. 5, 350-357[CrossRef][Medline] [Order article via Infotrieve] |
12. | Fox, J. A. (1995) Pfleugers Arch. 429, 873-875[Medline] [Order article via Infotrieve] |
13. | Boland, L. M., and Bean, B. P. (1993) J. Neurosci. 13, 516-533[Abstract] |
14. | Boland, L. M., Morrill, J. A., and Bean, B. P. (1994) J. Neurosci. 14, 5011-5027[Abstract] |
15. | Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., McCue, A. F., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Science 257, 389-395[Medline] [Order article via Infotrieve] |
16. | Ellinor, P. T., Zhang, J. F., Horne, W. A., and Tsien, R. W. (1994) Nature 372, 272-275[CrossRef][Medline] [Order article via Infotrieve] |
17. | McDonough, S. I., Swartz, K. J., Mintz, I. M., Boland, L. M., and Bean, B. P. (1996) J. Neurosci. 15, 2612-2623 |
18. |
Feng, Z. P.,
Hamid, J.,
Doering, C.,
Jarvis, S. E.,
Bosey, G. M.,
Bourinet, E.,
Snutch, T. P.,
and Zamponi, G. W.
(2001)
J. Biol. Chem.
276,
5726-5730 |
19. |
Beedle, A. M.,
and Zamponi, G. W.
(2000)
Biophys. J.
79,
260-270 |
20. | Catterall, W. A. (2000) Annu. Rev. Cell Dev. Biol. 16, 521-555[CrossRef][Medline] [Order article via Infotrieve] |
21. | Adams, M. E., Myers, R. A., Imperial, J. S., and Olivera, B. M. (1993) Biochemistry 30, 12566-12570 |
22. | Mintz, I. M., Venema, V. J., Swiderek, K. M., Lee, T. D., Bean, B. P., and Adams, M. E. (1992) Nature 27, 827-829 |
23. | McDonough, S. I., Mintz, I. M., and Bean, B. P. (1997) Biophys. J. 72, 2117-2128[Abstract] |
24. | Lampe, R. A., Defeo, P. A., Davison, M. D., Young, J., Herman, J. L., Spreen, R. C., Horn, M. B., Mangano, T. J., and Keith, R. A. (1993) Mol. Pharmacol. 44, 451-460[Abstract] |
25. | Piser, T. M., Lampe, R. A., Keith, R. A., and Thayer, S. A. (1995) Mol. Pharmacol. 48, 131-139[Abstract] |
26. |
McDonough, S. I.,
Lampe, R. A.,
Keith, R. A.,
and Bean, B. P.
(1997)
Mol. Pharmacol.
52,
1095-1104 |
27. | Ertel, E. A., Warren, V. A., Adams, M. E., Griffin, P. R., Cohen, C. J., and Smith, M. M. (1994) Biochemistry 33, 5098-5108[Medline] [Order article via Infotrieve] |
28. | Mintz, I. M. (1994) J. Neurosci. 14, 2844-2853[Abstract] |
29. | Miller, C. (1995) Neuron 15, 5-10[Medline] [Order article via Infotrieve] |
30. |
Chahine, M.,
Sirois, J.,
Marcotte, P.,
Chen, L.,
and Kallen, R. G.
(1998)
Biophys. J.
75,
236-246 |
31. | Li, R. A., Tsushima, R. G., Kallen, R. G., and Backx, P. H. (1997) Biophys. J. 73, 1874-1884[Abstract] |
32. |
Li, R. A.,
Ennis, I. L.,
Velez, P.,
Tomaselli, G. F.,
and Marban, E.
(2000)
J. Biol. Chem.
275,
27551-27558 |
33. | Stea, A., Soong, T. W., and Snutch, T. P. (1995) in Handbook of Receptors and Channels: Ligand- and Voltage-gated Ion Channels (North, R. A., ed) , pp. 113-141, CRC Press Inc., Boca Raton, FL |
34. | Olivera, B. M., Gray, W. R., Zeikus, R., McIntosh, J. M., Varga, J., Rivier, J., de Santos, V., and Cruz, L. J. (1985) Science 230, 1338-1343[Medline] [Order article via Infotrieve] |
35. | Ridgeway, B., Wallace, M., and Gerayli, A. (2000) Pain 85, 287-289[CrossRef][Medline] [Order article via Infotrieve] |
36. | Penn, R. D., and Paice, J. A. (2000) Pain 85, 291-296[CrossRef][Medline] [Order article via Infotrieve] |