From the Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia
Received for publication, December 14, 2000, and in revised form, January 9, 2001
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ABSTRACT |
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The effects of the antihelmintic,
ivermectin, were investigated in recombinantly expressed human
Ivermectin (22,23-dihydroavermectin B1a) is
macrocyclic lactone widely used as an antiparasitic agent in domestic
animals and is considered the drug of choice for lymphatic filariasis and river blindness (onchocerciasis) in humans (1-3). The target of
its antiparasitic action is believed to be an ivermectin-sensitive glutamate-gated Cl The GluClR and the GABAAR belong to the ligand-gated ion
channel superfamily, which also includes the nicotinic acetylcholine receptor cation channel (nAchR), the serotonin type 3 receptor cation
channel, and the glycine receptor chloride channel (GlyR) (4, 11-14).
The mechanisms of action of ivermectin and its analogues have been
investigated in several members of this family. For example, ivermectin
irreversibly activates the GluClR over a concentration range from 0.1 to 1 µM, although at lower concentrations (<0.01 µM) it induces potentiation of glutamate-gated currents
(4, 5). The effects of ivermectin on the GABAAR also
include potentiation of GABA-gated currents (10, 15, 16) as well as
direct, reversible receptor activation (10, 17, 18), although both
effects have not always been observed in some preparations. In
addition, ivermectin has been shown to potentiate
acetylcholine-mediated responses in the recombinantly expressed
The effects of ivermectin on a variety of members of the ligand-gated
ion channel superfamily prompted us to test its actions on
recombinantly expressed human Mutagenesis and Expression of GlyR cDNAs--
The human GlyR
Electrophysiology--
The cells were observed using a
fluorescent microscope, and currents were measured using the whole cell
patch clamp configuration. Cells were perfused by a control solution
that contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, with the
pH adjusted to 7.4 with NaOH. Patch pipettes were fabricated from
borosilicate hematocrit tubing (Vitrex, Modulohm, Denmark) and
heat-polished. Pipettes had a tip resistance of 1.5-3 M
Ivermectin (Sigma) was stored frozen as a 10 mM stock
solution in dimethyl sulfoxide for up to 2 weeks. When dissolved into the perfusion solution, the final concentration of dimethyl sulfoxide was no more than 0.3%. Strychnine, picrotoxin, and zinc were also prepared from frozen stocks at concentrations of 10 mM (in
water), 100 mM (in dimethyl sulfoxide), and 100 mM (in water), respectively.
The effects of ivermectin were tested with the following procedure.
First, the glycine dose-response was measured by applying increasing
concentrations of glycine at 30-s intervals. Then two brief
applications of glycine at the half-saturating concentration (EC50) were followed by two brief applications at a
saturating concentration (10 × EC50), all at 30-s
intervals. Provided the current amplitude remained constant, the
averaged current amplitudes were used as the control. Following this,
ivermectin was applied until a steady-state response was attained
(usually <1 min). Because ivermectin induced irreversible activation,
only a single ivermectin treatment was obtained per cell, and the
coverslip was discarded after each recording.
Data Analysis--
Glycine dose responses were measured by
applying a series of glycine concentrations to each cell. Glycine
dose-response parameters were quantitated by fitting the Hill equation
to individual dose responses by a nonlinear curve fitting routine
(Origin 4.0, Northampton, MA). The EC50 and Hill
coefficient (nH) values thus obtained were then
averaged as means ± S.E. The irreversible nature of ivermectin activation meant that only one concentration could be applied on one
cell. In this case, current amplitude was normalized to the saturating
(10 × EC50) glycine-induced current in the same cell
and then averaged with the data recorded from other cells at the same
concentration. The pooled results recorded at different concentrations
from different cells were then fitted with the Hill equation to obtain
the Imax/Imax(Gly),
EC50, and Hill coefficient (nH)
values. The nH values obtained in such a manner
must be interpreted with caution as curve fits to averaged dose
responses typically underestimate its true value. Because of this
uncertainty, the present study avoids drawing inferences from
nH data. Where possible, statistical
significance was determined by one-way ANOVA, with p < 0.05 representing significance. However, because only a single EC50 value was obtained for the pooled ivermectin dose
responses, a simple one-way ANOVA could not always be performed. In
such cases the statistical significance of differences in ivermectin responses between wild-type and mutant GlyRs was determined with a
general linear model of two-way analysis of variance (ANOVA) (Minitab
13.20, State College, PA), with the two factors being ivermectin
concentration and phenotype, with p < 0.01 representing significance. The p values thus obtained using
this analysis are shown in Table II.
Ivermectin Activation of the GlyR--
Examples of the effects of
ivermectin on wild-type (WT)
The ivermectin dose-response relationship was constructed by
normalizing the magnitude of the current activated by a known concentration of ivermectin to the magnitude of the current activated by a saturating concentration (10 × EC50) of glycine
in the same cell. Thus, a single point on a dose-response curve was
obtained from each cell. By measuring the relative magnitudes of
ivermectin-gated currents at a single concentration in many cells, a
pooled ivermectin dose response was compiled as shown in Fig.
2A. Each point in this figure
was averaged from at least three different cells. The curve, which was
fit to all points, had an EC50 of 0.39 µM and
an nH of 0.59. The ivermectin EC50
and nH values for all other GlyR constructs
investigated in this study were calculated in the same way, and all
results are summarized in Table I. The respective glycine EC50 and nH
values for the same GlyR constructs were calculated from entire dose
responses measured in individual cells, and these results are also
summarized in Table I.
GlyRs in vivo are considered to exist as heteromers
comprising
We then investigated the ivermectin current-voltage (I-V) relationship
of the WT GlyR according to the following procedure. First, a
saturating concentration (0.25 mM) of glycine was applied at Ivermectin Potentiation of Glycine-gated Currents--
As shown in
Fig. 1 (top panel), low concentrations (0.03 µM) of ivermectin potentiated glycine-gated currents.
Both the glycine and ivermectin dependence of this effect were
investigated. As shown in the example in Fig.
3A, 0.03 µM
ivermectin dramatically increased the magnitude of current activated by
a 10 µM (EC5) concentration of glycine
(upper panel) but appeared to slightly diminish the
amplitude of currents activated by a 250 µM (saturating) glycine concentration (lower panel). The magnitude of the
potentiation induced by 0.03 µM ivermectin was measured
at the following glycine concentrations (with approximate EC values in
parentheses): 10 (EC5), 25 (EC50), 30 (EC60), 100 (EC95), and 250 µM
(EC100). The mean percentage change in glycine-gated
current at each concentration is summarized in Fig. 3B.
Since ivermectin had no significant effect at the EC60
glycine concentration, it is not possible to conclude that it acted by
a simple enhancement of the apparent glycine affinity. The results
suggest instead that ivermectin selectively potentiated currents
activated by the lowest glycine concentrations, without dramatically
changing the glycine EC50 value. The significant reduction
in current magnitude at 250 µM glycine is expected since
ivermectin is a partial agonist.
The range of ivermectin concentrations that induced potentiation of
glycine currents was very narrow. In fact, 0.01 µM
ivermectin induced no significant potentiation of glycine responses nor
did it induce significant direct current activation (n = 4 cells). On the other hand, 0.1 µM ivermectin directly
activated 27 ± 5% (n = 4 cells) of the
saturating glycine-gated current but also induced no significant
potentiation of glycine-gated currents (n = 4 cells).
At a concentration of 0.03 µM, ivermectin exhibited a
dual effect as a potentiator and an activator inducing 7.8 ± 1.5% (n = 4 cells) of the saturating glycine-gated current.
Pharmacology of Ivermectin-gated Currents--
The pharmacological
profile of ivermectin-gated currents was investigated by measuring the
inhibitory potencies of strychnine, picrotoxin, and zinc. Strychnine is
considered to act as a classical competitive antagonist of glycine with
an affinity in the nanomolar range (25-27). As shown in Fig.
4A (left panel), 10 µM strychnine strongly inhibited the current activated by
a saturating (250 µM) concentration of glycine. However,
10 µM strychnine had no significant inhibitory effect on
the current activated by a saturating (3 µM)
concentration of ivermectin. The effect of strychnine was also
investigated at the lower ivermectin concentration of 0.3 µM, where it was found to induce significant inhibition.
The effect of 10 µM strychnine on glycine- and
ivermectin-gated currents are summarized in Table
II. The results indicate that
ivermectin-gated currents have a dramatically reduced sensitivity to
strychnine.
Since ivermectin is an irreversible agonist, the reduction in
strychnine efficacy could have resulted from the bound ivermectin sterically hindering strychnine from accessing its binding site. To
investigate this possibility, we applied strychnine first and sought to
determine whether pre-bound strychnine could inhibit the subsequent
rate of activation of ivermectin-induced currents. The time constant of
strychnine unbinding was estimated as indicated in Fig. 4B,
left panel. Following removal of strychnine, the saturating current magnitude was regularly monitored by brief applications of 250 µM glycine. Fitting first-order exponential curves to the current peaks enabled us to estimate a mean unbinding time constant for
10 µM strychnine of 5.2 ± 0.26 s
(n = 5) and for 100 µM strychnine of
22.1 ± 1.4 s (n = 5). The mean time constant
of current activation by 3 µM ivermectin was 4.4 ± 1.0 s (n = 6). This value was not significantly
different to the unbinding time constant of 10 µM strychnine (one-way ANOVA, p > 0.05) but was
significantly faster than the unbinding time constant of 100 µM strychnine (p < 0.05). Therefore, if
strychnine and ivermectin are competing for a common or overlapping
binding site, the dissociation rate of 100 µM strychnine should be the rate-limiting step in the activation of ivermectin-gated currents. An example of an experiment designed to investigate this
possibility is shown in Fig. 4B, right panel.
When 3 µM ivermectin was applied immediately after a long
(30 s) application of 100 µM strychnine, the mean current
activation time constant was 2.9 ± 0.72 s (n = 3). This value was not significantly different to the activation
time constant in the absence of strychnine (one-way ANOVA,
p > 0.05), indicating that pre-bound strychnine has no effect on the activation rate of ivermectin-gated currents. Therefore, in contrast to its effect on glycine-gated currents, strychnine is a
relatively weak antagonist of ivermectin-gated currents.
Although the mechanism of action of picrotoxin on the GlyR has not been
unequivocally resolved, evidence to date indicates that it acts as an
allosteric inhibitor (23, 28). As shown in Fig. 4C
(left panel), 1 mM picrotoxin strongly inhibited
currents activated by 250 µM glycine. However, in the
same cell it was apparent that the same picrotoxin concentration had
little effect on currents activated by 3 µM ivermectin
(Fig. 4C, right panel). The inhibitory effects of
picrotoxin on both glycine- and ivermectin-gated currents are
summarized in Table II. The results indicate that picrotoxin is a
comparatively poor antagonist of ivermectin-activated currents.
Zinc acts as an allosteric potentiator of glycine-gated currents at low
(<3 µM) concentrations and as a competitive antagonist at higher concentrations (29, 30). As shown in Fig. 4D, 1 mM zinc strongly inhibited glycine-gated currents but had
little effect on ivermectin-gated currents. The averaged results
displayed in Table II confirm that zinc antagonism of ivermectin-gated
currents is also very weak. Taken together, these results indicate that ivermectin-gated currents are pharmacologically distinct from glycine-gated currents.
Effects of Glycine-binding Site Mutations on Ivermectin-gated
Currents--
Previous studies have revealed that two regions in the
GlyR Effect of a Startle Disease Mutation on Ivermectin
Efficacy--
R271Q is a heritable mutation in the human GlyR
As indicated above, prior application of 3 µM ivermectin
induced glycine to irreversibly increase the current magnitude. It is
noteworthy that this irreversible "glycine-enhanced" current was
much larger in magnitude than that induced by a saturating glycine
concentration prior to ivermectin exposure (Fig. 5B). In
fact, the magnitude of current activated by 7 mM
(EC50) glycine was increased by 5.8 ± 2.1 (n = 4) times after exposure to 3 µM ivermectin. The pharmacology of this glycine-enhanced current was
investigated by testing the effect of picrotoxin. In the WT GlyR,
picrotoxin acts as a competitive antagonist of glycine-gated currents
(28). However, in the R271Q mutant GlyR, picrotoxin is converted into a
noncompetitive inhibitor of glycine-gated currents with an
IC50 of 5 µM (28). When applied at a
concentration of 1 mM, picrotoxin inhibited the
irreversible glycine-enhanced current by 15 ± 7%
(n = 4), indicating that this current is dramatically less picrotoxin-sensitive than the glycine-gated current. Given the
weak picrotoxin sensitivity and the irreversible activation, this
current more closely resembles the ivermectin-gated current than the
glycine-gated current. The glycine exposure reduced the mean ivermectin
EC50 value from 7.9 to 6.6 µM. These
observations support the conclusion that a short application of glycine
significantly increases the efficacy with which the pre-bound
ivermectin can irreversibly activate the channels.
Effect of a Fast Desensitization Mutant on Ivermectin-gated
Responses--
A final series of experiments was directed at
determining whether a prolonged glycine exposure could induce
cross-desensitization of ivermectin-gated currents. This experiment is
difficult to perform using recombinantly expressed WT GlyR
In the WT GlyR, a saturating concentration (30 µM) of
ivermectin induced a current that decayed at approximately the same rate as the glycine-gated current recorded in the same cell (Fig. 6A). Furthermore, when
ivermectin was coapplied with glycine, the rate of current decay was
not changed (Fig. 6B). Similar results were observed in four
cells. As discussed below, these results contrast dramatically with
those recorded from the I244A mutant GlyR.
Apart from its effects on desensitization, I244A also reduced both the
glycine sensitivity and the agonist efficacy of
As displayed in Fig. 6D, ivermectin potently activated the
I244A mutant GlyR after the receptor was completely desensitized to
glycine. The mean ratio of the saturating ivermectin- versus glycine-gated current after glycine-induced desensitization was 1.3 ± 0.2 (n = 4), which using a one-way ANOVA
was not significantly different from that obtained before
glycine-induced desensitization (p > 0.05). The rate
of ivermectin-induced channel activation was not affected by the
presence of glycine. The mean activation time constant of currents
gated by 30 µM ivermectin in the presence of glycine was
5.3 ± 2.0 s (n = 4), whereas in the absence
of glycine was 3.6 ± 0.6 s (n = 3). By using
a one-way ANOVA, these values are not significantly different
(p > 0.05). These results demonstrate that ivermectin
activates the I244A mutant GlyR after it has been completely
desensitized to a prior glycine application. Furthermore, the
ivermectin activation rate is not changed in the glycine-induced
desensitized state.
Novel Mechanism of Ivermectin Activation--
Ivermectin exerted
dual effects on the WT GlyR. At low (0.03 µM)
concentrations it potentiated the response to sub-saturating glycine
concentrations, and at higher (
Ivermectin also exerts a range of effects on some other members of the
ligand-gated ion channel superfamily. For example, it reversibly
activates recombinantly expressed
The present study demonstrates that ivermectin-gated currents are
relatively insensitive to picrotoxin (Table II). This contrasts with
the effects of picrotoxin on the GluClR, where the picrotoxin sensitivity was similar for ivermectin- and glutamate-gated currents (Table III). The present study also found that the ivermectin-gated currents exhibited a dramatically reduced sensitivity to strychnine and
zinc (Table II). The reduction in strychnine potency could not be
explained by ivermectin preventing the access of strychnine to its
binding site. The novel pharmacology of the ivermectin-induced activated state supports the conclusion that ivermectin activates the
GlyR by a novel mechanism. In the
In addition to activating the channels by a novel mechanism, it is
likely that ivermectin binds to a novel, as yet unidentified, binding
site. This study has shown that the mutation of known glycine-binding
sites had very little effect on ivermectin sensitivity (Table I). In
particular, the Y202F and T204A mutations, which strongly disrupted the
apparent glycine affinity, had little effect on ivermectin sensitivity.
Thus, although the ivermectin- and glycine-binding sites may overlap to
some degree, the two molecules clearly do not bind to an identical set
of contact sites. Since Tyr202 is both a glycine- and
strychnine-binding site (26, 27), the observation that ivermectin does
not bind to Tyr202 may explain the reduced strychnine
sensitivity of the ivermectin-gated response.
Further evidence for a novel mechanism of action by ivermectin was
sought by investigating whether it could activate a GlyR that had
already been completely desensitized to glycine. As displayed in Fig.
6D, ivermectin strongly and irreversibly activated the I244A
mutant GlyR after it had previously been completely desensitized to
glycine, suggesting that it could return desensitized channels directly
to the activated state. This strongly supports the previous conclusion
that ivermectin activates the GlyR via a novel mechanism.
Allosteric Disruption of the Ivermectin Activation
Mechanism--
The mechanism of action of ivermectin was investigated
further by examining the effect of the R271Q mutation. This mutation, which is a cause of human startle disease, has been shown to reduce the
apparent affinities of the agonists glycine,
The I244A mutation causes relatively small reductions in the apparent
agonist affinities of glycine, Conclusion--
This study concludes that ivermectin-gated
currents in the GlyR are similar to those in some GluClRs in that they
potentiate agonist responses at low concentrations and irreversibly
activate the receptor at higher concentrations. However, the GlyR
differs in that the ivermectin-gated currents have a different
pharmacology to glycine-gated currents. Consistent with this
observation, this study demonstrates that the ivermectin- and
glycine-binding sites are not identical. In addition, the observation
that ivermectin potently activates a mutant GlyR after its response to
glycine is completely desensitized provides strong evidence for a novel mechanism of activation. Furthermore, mutations that are known to
disrupt the agonist signal transduction mechanism have a relatively small effect on the ivermectin agonist transduction mechanism relative
to those of glycine, 1 homomeric and
1
heteromeric glycine receptors (GlyRs). At low (0.03 µM)
concentrations ivermectin potentiated the response to sub-saturating
glycine concentrations, and at higher (
0.03 µM)
concentrations it irreversibly activated both
1
homomeric and
1
heteromeric GlyRs. Relative to
glycine-gated currents, ivermectin-gated currents exhibited a
dramatically reduced sensitivity to inhibition by strychnine,
picrotoxin, and zinc. The insensitivity to strychnine could not be
explained by ivermectin preventing the access of strychnine to its
binding site. Furthermore, the elimination of a known glycine- and
strychnine-binding site by site-directed mutagenesis had little effect
on ivermectin sensitivity, demonstrating that the ivermectin- and
glycine-binding sites were not identical. Ivermectin strongly and
irreversibly activated a fast-desensitizing mutant GlyR after it had
been completely desensitized by a saturating concentration of glycine.
Finally, a mutation known to impair dramatically the glycine signal
transduction mechanism had little effect on the apparent affinity or
efficacy of ivermectin. Together, these findings indicate that
ivermectin activates the GlyR by a novel mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel receptor
(GluClR)1 that exists in a
number of invertebrate phyla (4-7). Ivermectin also acts as an
anticonvulsant in a variety of vertebrate seizure models (8-10).
However, GluClRs have not been demonstrated to exist in vertebrates,
and the anticonvulsant actions of ivermectin in a mouse seizure model
has recently been shown to be mediated by GABA type A receptors
(GABAARs) (10).
7 nAChR (19). Finally, ivermectin has long been known to
displace [3H]strychnine in radiolabeled binding studies
(20), implying that it may exert some effect on the GlyR. Indeed,
ivermectin has recently been demonstrated to act as a
use-dependent inhibitor of a GlyR that is endogenously
expressed in primary cultured rat cortical neurons (10).
1 homomeric and
1
heteromeric GlyRs. We find that ivermectin acts as
an allosteric potentiator of glycine-gated currents at low (0.03 µM) concentrations and as a potent, irreversible agonist
at higher concentrations. In addition, since ivermectin affinity is not
affected by mutations to a well characterized glycine binding domain,
and the pharmacology of ivermectin-gated currents is different to that
of glycine-gated currents, it appears that ivermectin acts via a novel
mechanism to activate the GlyR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
subunit cDNAs were subcloned into the
pCIS2 and pIRES2-EGFP plasmid vectors (CLONTECH,
Palo Alto, CA), respectively. Because GlyR
subunits can efficiently
assemble into functional GlyRs as either
homomers or
/
heteromers, green fluorescent protein expression was used as an
indicator of GlyR
subunit expression. Site-directed mutagenesis was
performed using the QuickChange mutagenesis kit (Stratagene, La Jolla,
CA), and the successful incorporation of mutations was confirmed by sequencing the clones. Adenovirus-transformed human embryonic kidney
293 cells (ATCC CRL 1573) were passaged in minimum essential medium
supplemented with 2 mM glutamate, 10% fetal calf serum, and the antibiotics penicillin 50 IU/ml and streptomycin 50 µg/ml (Life Technologies, Inc.). Cells were transfected using a calcium phosphate precipitation protocol (21). When cotransfecting the GlyR
and
subunits, their respective cDNAs were combined in a ratio
of 1:10 (22). After exposure to transfection solution for 24 h,
cells were washed twice using the culture medium and used for recording
over the following 24-72 h.
when filled
with the standard pipette solution which contained (in mM)
145 CsCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA,
with the pH adjusted to 7.4 with NaOH. After establishment of the whole
cell configuration, cells were voltage-clamped at
40 mV, and membrane currents were recorded using an Axopatch 1D amplifier and pCLAMP7 software (Axon Instruments, Foster City, CA). The cells were perfused by a parallel array of microtubular barrels through with solutions were
gravity-induced. The amplifier series resistance compensation was used
to compensate for at least 50% of the series resistance error.
Experiments were conducted at room temperature (18-22 °C).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 homomeric GlyRs are shown
in Fig. 1. It can be seen that
ivermectin, at concentrations of 0.3 µM or greater,
induced irreversible channel activation. Such effects were not reversed
by a 10-min wash in ivermectin-free control solution. Because of its
irreversible effect, only one ivermectin application could be recorded
from each cell. Hence, the traces in Fig. 1 corresponding to different ivermectin concentrations were recorded from different cells, although
both traces in any given row were recorded from the same cell. It is
notable that the ivermectin-induced activation was much slower than
that induced by glycine, particularly at the lower ivermectin
concentrations. Although a 0.03 µM application of
ivermectin activated little current, it did result in a strong potentiation of the current activated by a sub-saturating glycine concentration (Fig. 1, top panel). This effect is considered
further below. Ivermectin was a partial agonist of the GlyR, with a
saturating (30 µM) concentration activating 77 ± 6% (n = 6) of the current activated by a saturating
(250 µM) concentration of glycine. When ivermectin was
applied at a saturating concentration, glycine activated no additional
current (Fig. 1, bottom panel), indicating that ivermectin
and glycine compete for the activation of the same population of
GlyRs.
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Fig. 1.
Ivermectin potentiation and activation of the
GlyR. In this and all subsequent figures, all displayed traces
were recorded from cells expressing WT or mutated 1
homomeric GlyRs using whole cell recording. In this figure, traces in
different rows were obtained from different cells, although both traces
on a single row were from the same cell. Each cell was treated first
with two applications of a half-saturating (25 µM)
glycine concentration, followed by two applications of a saturating
(250 µM) glycine concentrations (left panels).
Following this, ivermectin was applied at the indicated concentration,
and glycine responses were recorded again (right
panels).
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Fig. 2.
Properties of ivermectin-gated currents.
A, the ivermectin dose-response curve was compiled as
described in the text. The curve was fitted with an
EC50 of 0.39 µM and an
nH of 0.59. B, current-voltage
relationship curves for glycine- and ivermectin-gated currents were
determined as described in the text. Current amplitude was normalized
to the saturating concentration (250 µM) glycine-induced
current recorded at 40 mV. The reversal potential was
0.2 mV for
glycine and 3.7 mV for ivermectin.
Summary of glycine and ivermectin effects on WT and mutant GlyRs
homomeric GlyRs.
and
subunits in the ratio 3:2 (23). Since the
1/
heteromeric GlyR differs pharmacologically from
the
1 homomeric GlyR (22, 24), it was possible that the
heteromers may respond differently to ivermectin. However, the presence
of the
subunit had no significant effect on ivermectin sensitivity
(Table I). Hence, all experiments described in the remainder of this
paper were performed on
1 homomeric GlyRs.
40 mV, followed by a repeated glycine application and a 3 µM (saturating) ivermectin application at a specific
holding potential of
70,
40,
20, 0, 20, or 40 mV. The glycine-
and ivermectin-induced currents measured at the specific holding
potential were normalized to the initial glycine current recorded at
40 mV to generate the I-V curve displayed in Fig. 2B. This
figure demonstrates that, in contrast to the linear I-V relationship
that characterized glycine-gated currents, the ivermectin-gated
currents displayed weak outward rectification. Differences in
rectification between ivermectin- and glutamate-gated conductances have
also been observed in the GluClR (4). The reversal potentials for
currents activated by both glycine (
0.2 mV) and ivermectin (+3.7 mV)
approximated to the equilibrium potential (0 mV) for chloride ions.
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Fig. 3.
Ivermectin potentiation of submaximal
glycine-gated currents. A, pre-applied 0.03 µM ivermectin potentiated the currents activated by a 10 µM (EC5) glycine concentration (upper
trace) and slightly reduced the currents activated by a 250 µM (saturating) glycine-induced current (lower
trace). Both traces were from different cells. B,
glycine concentration dependence of potentiation by 0.03 µM ivermectin. All values were averaged from at least
three cells.
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Fig. 4.
Pharmacology of ivermectin-gated
currents. A, a 10 µM concentration of
strychnine strongly inhibits currents activated by a saturating (250 µM) concentration of glycine (left panel) but
has little effect on the current activated by a saturating (3 µM) concentration of ivermectin (right panel).
B, the time course of recovery from an application of 100 µM strychnine is slow and can be quantitated by regularly
monitoring current with short applications of 250 µM
glycine (left panel). However, a pre-application of 100 µM strychnine has little apparent effect on the rate of
activation of ivermectin-mediated currents in the same cell
(right panel). C, a 1 mM
concentration of picrotoxin strongly inhibits currents activated by a
saturating (250 µM) concentration of glycine (left
panel) but has little effect on the current activated by a
saturating (3 µM) concentration of ivermectin
(right panel). D, similarly, 1 mM
zinc strongly inhibits currents activated by a saturating (250 µM) concentration of glycine (left panel) but
has little effect on the current activated by a saturating (3 µM) concentration of ivermectin (right
panel).
Percentage inhibition by picrotoxin, strychnine, and zinc of glycine-
and ivermectin-induced currents in the WT GlyR
1 subunit external N-terminal domain,
Phe159-Gly160-Tyr161 and
Lys200-Tyr202-Thr204, are major
determinants of the glycine- and strychnine-binding sites (26, 27,
31-33). Accordingly, the effects of ivermectin were investigated on
the F159Y, Y161F, Y202F, and T202A mutant GlyRs. Since ivermectin
remained an irreversible agonist of all these mutant GlyRs, the
ivermectin dose responses were measured as described in Fig.
2A. The glycine dose responses for each mutant GlyR were
also measured, and all results are summarized in Table I. Consistent
with previous studies (26, 27, 31-33), the glycine sensitivity was
dramatically reduced (>200-fold) by the Y202F and T204A mutations,
although the sensitivity was modestly increased by the F159Y and Y161F
mutations. However, the ivermectin sensitivity was only weakly affected
(1.4-6.9-fold) by any of these mutations (Table I). In addition, 0.03 µM ivermectin also potentiated half-saturating concentration glycine-induced current in each of the 4 mutant GlyRs
(not shown). Thus, ivermectin does not act by binding to the
glycine-binding site formed by Tyr202 and
Thr204.
1 subunit that underlies familial startle disease (34).
This mutation has previously been shown to induce a large reduction in
both glycine sensitivity and single channel conductance (35, 36). In
addition, this mutation also converts the glycinergic agonists,
-alanine and taurine, from agonists into competitive glycine
antagonists (37, 38). The effects of this mutation are most likely to be mediated by a disruption in the signal transduction process linking
the ligand-binding sites to the channel activation gate (38, 39). Given
these findings, we hypothesized that the R271Q mutation may also affect
the efficacy with which ivermectin is able to activate the channel.
Although 0.03 µM ivermectin induced little direct
activation of the R271Q mutant GlyR, it potentiated the magnitude of
currents activated by a 7 mM (EC50)
concentration of glycine (Fig.
5A). Since this is similar to
the effect of ivermectin on the WT GlyR, the mutation has apparently
not altered the receptor sensitivity to low ivermectin concentrations.
On the other hand, although the WT GlyR is strongly activated by 3 µM ivermectin, this concentration induced only a weak
direct activation of the R271Q mutant GlyR (Fig. 5B). In
addition, we were surprised to find that prior exposure of GlyRs to 3 µM ivermectin converted glycine into an irreversible
agonist (Fig. 5B), an effect that is considered further
below. When applied at a concentration of 30 µM, the
ivermectin-gated current was 4.8 ± 1.2 (n = 4)
times the magnitude of the current activated by a saturating (70 mM) glycine concentration (e.g. Fig.
5C). Once currents were maximally activated by ivermectin, a
subsequent application of 7 mM glycine induced an
additional small irreversible current (Fig. 5C). The mean
glycine and ivermectin EC50 and nH
values for the R271Q mutant GlyR are given in Table I. The results
indicate that although this mutation decreased the glycine sensitivity
by a factor of 273, the ivermectin sensitivity was decreased only by a
factor of 20. Taken together, these observations suggest that R271Q
disrupted the glycine gating mechanism to a much greater extent than
that of ivermectin. By comparison, it is relevant to note that the binding site mutations, Y202F and T202A, had no significant effect on
the relative magnitude of ivermectin- versus glycine-gated currents (Table I), although their glycine sensitivity was similar to
that of R271Q.
View larger version (9K):
[in a new window]
Fig. 5.
Effects of ivermectin on the R271Q mutant
GlyR. A-C, each cell was treated first with two
applications of a half-saturating (7 mM) glycine
concentration, followed by two applications of a saturating (70 mM) glycine concentration (left panels).
Following this, ivermectin was applied at the indicated concentration,
and glycine responses were recorded again (right panels).
Note the potent irreversible activation by 7 mM glycine in
B (right panel).
1 subunits because these display a slow rate of
desensitization. Indeed, most of the observed decay of glycine-gated
currents in whole cell recordings is due to chloride shift effects
(38). However, since glycine-gated currents in the I244A mutant GlyR
desensitize rapidly with a time constant of about 1 s (39), this
mutant receptor provides a suitable model for investigating the effects
of glycine-induced desensitization on ivermectin-gated currents.
View larger version (13K):
[in a new window]
Fig. 6.
Comparison of the effects of ivermectin on
the WT and I244A mutant GlyRs. Traces in both A and
B were recorded from WT GlyRs, whereas traces in
C and D were recorded from I244A GlyRs.
A, examples of WT GlyR currents activated by the indicated
(saturating) concentrations of glycine and ivermectin in the same cell.
B, ivermectin (30 µM) has little additional
effect when applied to WT GlyRs already maximally activated by 250 µM glycine. C, examples of I244A mutant GlyR
currents activated by the indicated (saturating) concentrations of
glycine and ivermectin. Both traces were recorded from the same cell.
Note the larger amplitude and the slower decay rate of the
ivermectin-gated current. D, a saturating concentration of
ivermectin is still able to irreversibly activate a large current even
after complete desensitization of the I244A mutant GlyR to a saturating
glycine concentration.
-alanine and taurine
relative to glycine (39). The glycine and ivermectin EC50
and nH values for the I244A mutant GlyR are
displayed in Table I. The I244A mutation reduces the glycine
sensitivity by a factor of 30, although the ivermectin sensitivity is
not affected. The recovery time constant from the glycine-induced fully
desensitized state to the resting closed state was measured to be
3.2 ± 0.49 s (n = 3). An example of a
glycine-gated current recorded from a cell expressing the I244A mutant
GlyR is displayed in Fig. 6C. The rapid rate of
desensitization of this current is apparent. An example of an
ivermectin-gated current recorded from the same cell 30 s later is
also shown (Fig. 6C). It is apparent that ivermectin not
only irreversibly activates this mutant GlyR but that the magnitude of
the current activated by a saturating (30 µM)
concentration of ivermectin is greater than that activated by a
saturating (8 mM) concentration of glycine. Indeed, the
ratio of the saturating ivermectin- versus glycine-gated
current averaged from four cells was 1.7 ± 0.2 (n = 4), suggesting that the I244A mutation reduces glycine efficacy. This
observation is consistent with a previous study that concluded that the
mutation disrupted the glycine signal transduction mechanism (39).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0.03 µM) concentrations it directly activated GlyR Cl
currents. These effects are
similar to those of ivermectin on the Caenorhabditis elegans
heteromeric GluClR and the Drosophila homomeric
GluClR (Table III). This functional
similarity may reflect the close phylogenetic relationship between the
GluClR and GlyR gene families (14). However, the results of the present study contrast with those recently found in GlyRs from cultured cortical neurons, where 1 µM ivermectin inhibited
glycine-activated currents in a use-dependent manner, while
simultaneously slowing the channel closing rate (10). Differences in
ivermectin effects may depend on subunit composition (Table III), and
since the subunit composition of the GlyR studied in (10) is undefined,
it is not possible at present to resolve these differences.
Comparison of glutamate- and ivermectin-PO4-gated currents in
various GluClRs
1
2,
1
1
2,
1
2
2,
1
2
2s,
1
3
2, and
1
2s GABAARs (10, 18) as well as a GABA-mediated Cl
current in dorsal root ganglion
neurons (17). Low ivermectin concentrations (<0.1 µM)
potentiate GABA-mediated Cl
currents in hippocampal
neurons (15), GABAARs expressed from chick brain mRNA
(16), as well as recombinantly expressed
1
1
2,
1
2
2, and
1
3
2 GABAARs
(10). Finally, in recombinantly expressed
7 homomeric
nAchRs, 30 µM ivermectin enhances the
acetylcholine-evoked current but displays no agonist activity (19). The
recently reported potentiating effect of ivermectin on P2X4
receptor channels (40) indicates that the effects of ivermectin are not
limited to members of the ligand-gated ion channel superfamily.
7 nAChR, ivermectin
potentiation also resulted in a modification of the receptor
pharmacological profile (19).
-alanine, and taurine
(37, 38). It also converts taurine and
-alanine into competitive
antagonists of glycine, without dramatically affecting their binding
affinities (38). These effects are consistent with a model whereby
R271Q completely disrupts the signal transduction pathway linking the
taurine and
-alanine agonist-binding sites to the activation gate
(39). Although the mutation appears to have a proportionately greater
disruptive effect on the agonist efficacy of the relatively lower
affinity agonists,
-alanine and taurine, it also reduces the agonist
efficacy of glycine (38, 39). This observation also suggests that since
ivermectin activates the GlyR with a higher affinity than does glycine,
the R271Q mutation should have a proportionately weaker disruptive
effect on the ivermectin agonist transduction mechanism. Indeed, by
demonstrating that the ivermectin sensitivity is little affected by
R271Q and that the maximum ivermectin-gated current is about 5 times
that activated by a saturating glycine concentration, this study
demonstrates that the glycine agonist transduction mechanism is
disrupted to a greater extent than that of ivermectin.
-alanine, and taurine and results in
-alanine and taurine being converted into partial agonists relative
to glycine (39). Thus, this mutation induces a similar, but relatively
weaker, phenotype than that induced by R271Q. Hence, it is not
surprising that the I244A mutation has little effect on ivermectin
sensitivity (Table I). Furthermore, since the mutation disrupted the
glycine signal transduction pathway to a relatively small extent (39),
it may be expected that the ratio of the peak ivermectin-gated current
to peak glycine-gated current would not be as large as it is for R271Q.
Indeed, the mean ratio for the I244A mutant GlyR was 1.7, whereas the
corresponding ratio for the R271Q mutant GlyR was 4.8 (Table I). Hence,
the results are consistent with the idea that I244A also disrupts the
ivermectin agonist transduction mechanism to a lesser extent than the
glycine transduction mechanism.
-alanine, or taurine. Together, these findings
indicate that ivermectin activates the GlyR by a novel mechanism.
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FOOTNOTES |
---|
* This work was supported in part by the Australian Research Council.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.
Supported by an Ernest Singer Postgraduate Scholarship from the
University of Queensland.
§ To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: 617-3365-3157; Fax: 617-3365-1766; E-mail: lynch@plpk.uq.edu.au.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M011264200
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ABBREVIATIONS |
---|
The abbreviations used are:
GluClR, glutamate-gated Cl channel receptor;
ANOVA, analysis of
variance;
GABA,
-aminobutyric acid;
GABAARs, GABA type A
receptors;
nAchR, nicotinic acetylcholine receptor;
GlyRs, glycine
receptors;
WT, wild-type.
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