|
|
|
|||||||
|
|||||||||
From the
Contrary to its effects on the
Glycine receptors (GlyRs)
The
dominant human neurological disorder, startle disease, is caused by
missense mutations that lead to the Arg at position 271 (Arg-271) of
the human GlyR
The
aim of the present study was to establish whether the actions of GlyR
antagonists may also be functionally coupled via the same residue. The
plant alkaloid, picrotoxin, is an antagonist of both GABA type A
receptors (GABA
Membrane currents were recorded using an Axopatch 1D amplifier and
PClamp software (Axon Instruments, Foster City, CA) from cells
voltage-clamped at -60 mV, corrected for liquid junction
potentials (26). The amplifier electronic series resistance
compensation was used to compensate for at least 50% of the series
resistance error. Experiments were performed at room temperature
(18-22 °C). Solution exchanges were performed using a
parallel array of microtubular barrels through which solutions were
gravity-fed into the recording chamber. Barrel mouths were positioned
under visual control to within 100 µm of target cells, and rapid
complete solution exchange was effected by moving the barrels with a
manually operated micromanipulator. For the experiments where a
constant rate of solution exchange was required for different pairs of
solutions (e.g. see Fig. 2, below), solutions were
always chosen to flow through the same pair of adjacently situated
barrels.
By using a similar
strategy as outlined in Fig. 2A, we examined whether
picrotoxin can bind efficiently in the absence of glycine. An example
of such an experiment is shown in Fig. 2B, where
preexposure of cells to 100 µM picrotoxin in the absence
of glycine was followed by the simultaneous removal of picrotoxin and
application of 1 mM glycine. As shown in
Fig. 2D, the time to half-activation was not
significantly different from the recovery time when picrotoxin was
co-applied with glycine. For the current traces displayed in
Fig. 2, A and B, this is illustrated by the
ability to superimpose all respective current activation segments in
Fig. 2C. Thus, picrotoxin binding is independent of the
presence of glycine.
In a second approach to check for possible use
dependence, we examined whether repeated glycine applications in the
constant presence of picrotoxin
(29) could cause a progressive
accumulation of inhibition. An example of an experiment designed to
test this is shown in Fig. 3A, where eight successive
2-s applications of glycine caused virtually no time-dependent
accumulation of inhibition. Pooled results from four cells, displayed
in Fig. 3B, confirm this trend. Hence, from these
experiments, it may be concluded that picrotoxin binding is not
dependent on the presence of glycine.
Inhibition of
GABA-gated currents by picrotoxin has been reported as being either
noncompetitive
(27, 31, 34, 35, 36, 37) or a mixture of competitive and noncompetitive
interactions
(28, 38, 39) . In the later
category, competitive interactions accounted for a relatively small
proportion of the overall inhibition. The present study demonstrates
that picrotoxin antagonism of GlyRs is purely competitive (
Fig. 4
and 5). This appears incompatible with the recent
observation
(40) that picrotoxin is a noncompetitive inhibitor
of glycine-gated currents in rat hypothalamic neurons. Since
differences in picrotoxin effects may depend on subunit composition
(20) and the subunit composition of the GlyRs studied in
(40) is undefined, it is not possible at present to resolve
these differences. As discussed below, since picrotoxin does not
displace glycine or strychnine binding, its competitive antagonism is
presumably mediated via an allosteric mechanism.
Picrotoxinin is
considerably more potent than picrotin in inhibiting the
GABA
There are at least two ways of
reconciling the results of Pribilla et al.(20) with
the observations presented here. Firstly, by mutating the Arg-271
transduction site, Pribilla et al. may have disrupted the
ability of picrotoxin to exert its antagonistic effect (see below).
Second, another of their mutated residues (Thr-265) is the homologue of
a residue in the nAChR that is involved in the conversion of an
antagonist-induced desensitized state into a conducting
state
(42) . Although originally proposed as a GABA
It should be considered whether the noncompetitive
inhibition by picrotoxin is due to channel block. By analogy with the
nAChR, the 271 residue forms part of the external channel vestibule. It
is possible that if a picrotoxin binding site were created at this site
by the mutation, then bound picrotoxin may be expected to occlude the
ion pore. If we suppose that such a blocking site accounts for the
observed noncompetitive antagonism, then, as a minimum, we are left
with the conclusion that the Arg-271 mutations dramatically modify the
coupling mechanism of a single class of extracellular picrotoxin
binding sites.
Like R271L and R271Q GlyRs, GABA
The
finding that Arg-271 is involved in the transduction of information
from both an agonist
(15) and an antagonist binding site (this
study) indicates that the allosteric pathways of both classes of
ligands converge at a common residue prior to the activation gate of
the channel. Thus, by simultaneously processing information from two
different binding sites, this residue may act as an integration point.
This may explain how antagonists, which act allosterically with
residues lining the channel pore, may competitively interact with
agonists. Conceivably, the transduction site itself could be the
functional site of action for some antagonists.
The values of the picrotoxin
half-inhibitory concentration (EC
All experiments were
performed using a glycine concentration of 1 mM.
The averaged proportionate inhibition and potentiation induced
by picrotin, picrotoxinin, and picrotoxin are shown for both R271L and
R271Q GlyRs. The conditions under which each was measured is given in
the text. Each antagonist was tested on each cell, and statistical
significance (indicated by *) was assessed using a paired t test, using a level of significance of p < 0.05.
We thank Vikki Falls, Cheryl Handford and Kerrie
Pierce for excellent technical assistance.
Volume 270,
Number 23,
Issue of June 9, pp. 13799-13806, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-aminobutyric acid type A
receptor, picrotoxin antagonism of the
1 subunit of the human
glycine receptor is shown to be competitive, not use-dependent, and
nonselective between the picrotoxin components, picrotin, and
picrotoxinin. Competitive antagonism and non-use dependence are
consistent with picrotoxin binding to a site in the extracellular
domain. The mutations Arg Leu or Arg
Gln at residue 271
of the glycine receptor
1 subunit, which are both associated with
human startle disease, have previously been demonstrated to disrupt the
transduction process between agonist binding and channel activation. We
show here that these mutations also transform picrotoxin from an
allosterically acting competitive antagonist to an allosteric
potentiator at low (0.01-3 µM) concentrations and to
a noncompetitive antagonist at higher (
3 µM)
concentrations. This demonstrates that arginine 271 is involved in the
transduction process between picrotoxin binding and its mechanism of
action. Thus, the allosteric transduction pathways of both agonists and
antagonists converge at a common residue prior to the activation gate
of the channel, suggesting that this residue may act as an integration
point for information from various extracellular ligand binding sites.
()
mediate
inhibitory neurotransmission in the spinal cord and
brainstem
(1) . Together with receptors for acetylcholine, GABA,
serotonin, and glutamate, they form the ligand-gated ion channel
receptor superfamily (2-6). Receptors of this class consist of
five membrane-embedded subunits arranged radially around a central
ion-conducting pore
(7) . Subunits of the different receptor
types share common structural components, including an N-terminal
extracellular domain, which contains the ligand-binding sites, and four
membrane-spanning domains (M1-M4), the second of which lines the
pore and contains residues responsible for channel activation,
desensitization, ionic selectivity, and the binding of noncompetitive
channel blockers
(8) . The ligand-binding sites and the channel
domain are distant from each other (7, 9, 10) and little is known about
the mechanism that links ligand binding to channel activation.
1 subunit being converted to either a Leu or a Gln
(11, 12). This residue lies at the extracellular border of the M2
domain, and these mutations caused a reduction in the magnitude of
glycine-gated currents by both decreasing glycine
sensitivity
(13, 14) and by redistributing
single-channel conductances toward lower unitary
levels
(14, 15) . They also converted taurine and
-alanine from agonists into competitive antagonists, indicating
that Arg-271 is a crucial residue in controlling agonist signal
transduction
(15) . The mutations of Arg-271 had no effect on the
binding of the competitive antagonist,
strychnine
(13, 14) , consistent with the earlier
proposal that it acts by steric hindrance of agonist
binding
(16, 17) . In the homologous nicotinic
acetylcholine receptor (nAchR), a series of antagonists has been
identified that ``competitively'' antagonizes agonist-induced
responses by interacting allosterically with residues within the M2
domain to stabilize a desensitized state
(18) . However, no
glycinergic antagonists have yet been shown to act in this way.
Rs) and GlyRs
(19) . A recent
study
(20) , which analyzed recombinant GlyRs mutated throughout
the M2 domain, concluded that picrotoxin bound to a site within the
channel pore and acted as a channel blocker. In this study, we
demonstrate that the action of picrotoxin on the WT GlyR is competitive
and displays no use dependence. These properties, which contrast
directly with those of picrotoxin on the GABAR, are not
readily reconciled with a binding site within the
pore
(8, 21) . We then show that the functional coupling
of picrotoxin is fundamentally altered by the mutations of Arg-271. The
results indicate that this residue is involved in the transduction of
information from a competitive antagonist binding site to at least one
domain controlling ion channel function of the GlyR.
Mutation and Expression of Human GlyR
Mutations in the cDNA encoding the human
GlyR Subunit cDNA subunit were constructed using the
oligonucleotide-directed polymerase chain reaction mutagenesis method
(22) and confirmed by sequencing the cDNA clones. Plasmid DNA encoding
both wild-type or mutated subunits was transiently
transfected into exponentially growing human embryonic kidney 293 cells
(ATCC CRL 1573) via the method of Chen and Okayama
(23) using
the vector pCIS2
(24) . Cells were cultured for 24 h prior to
transfection in Eagle's minimum essential medium in Hank's
salts, supplemented with 2 mM glutamine and 10% fetal calf
serum. After transfection for 24 h, the cells were washed twice, placed
in fresh culture medium, and used within 72 h.
Electrophysiology
Coverslips containing cultured
transfected cells were transferred into a small volume (2 ml) recording
chamber, which was continually perfused with a modified Ringer's
solution containing 140 mM NaCl, 5 mM KCl, 2
mM CaCl
, 1 mM MgCl, 10
mM Hepes, 10 mM glucose, pH 7.4, with NaOH.
Glycine-gated currents were measured using whole cell recording
techniques
(25) . Patch pipettes were fabricated from
borosilicate hemeatocrit tubing (Vitrex, Modulohm, Denmark) and had tip
resistances of 1-4 megaohms when filled with the standard
intracellular solution containing 145 mM CsCl, 2 mM
CaCl, 2 mM MgCl, 10 mM Hepes,
10 mM EGTA, pH 7.4, with CsOH. Picrotoxin, picrotoxinin, and
picrotin (Sigma) were dissolved in dimethyl sulfoxide and diluted with
distilled water to a 10 mM stock solution, with at a dimethyl
sulfoxide concentration of 5%. Stocks were stored frozen for up to 2
months, and freshly thawed aliquots were used on each experimental day.
Figure 2:
Rate of washout of picrotoxin-induced
inhibition is independent of the presence of glycine. All traces in
A-C are from the same cell. The hollowbars represent the application of 1 mM glycine and the
filledbars represent the application of 100
µM picrotoxin. Bars apply only to traces
indicated by filledcircles. Traces indicated by the
hollowcircles were produced in response to the
application of 1 mM glycine without any exposure to
picrotoxin and have been truncated for clarity. A, a 1.5-s
preexposure to glycine + picrotoxin results in relatively slowly
increasing current after washout of picrotoxin. Activation segments of
both traces are expanded in C. B, a 1.5-s preexposure to
picrotoxin immediately followed by glycine application also results in
a relatively slowly activating current. Activation segments of both
traces are expanded in C. C, when the time courses of currents
activated by glycine (hollowcircle) or following
washout of picrotoxin (filledcircle) are expanded,
normalized, and superimposed, it is clear that picrotoxin washout slows
the apparent activation rate of currents in a glycine-independent
manner. D, time to half-activation of currents activated by
glycine in the absence of picrotoxin preexposure, following a 1.5-s
preexposure to glycine plus picrotoxin, and following 1.5-s preexposure
to picrotoxin alone. Analysis of variance (degrees of freedom (d f ) = 2, 14; F-statistic (F) = 7.5; p = 0.008), and subsequent F-tests revealed that
preexposure to picrotoxin (df = 4; F =
16.2; p = 0.01) or picrotoxin and glycine (df = 4; F = 10.2; p = 0.02)
both increased half-activation times of currents. No difference,
however, was found between the half-activation times after preexposure
to picrotoxin alone and to picrotoxin plus glycine (df = 4; F = 1.6; p = 0.3).
Data are averaged from 5 cells, and errorbars are
S.E. Asterisks indicate p 
0.02 with respect to
no picrotoxin preexposure.
Action of Picrotoxin on the WT GlyR
The voltage
sensitivity of the picrotoxin-induced inhibition was examined using an
approximately half-inhibiting (30 µM) concentration of
picrotoxin in the presence of a saturating (1 mM)
concentration of glycine. An example of a typical experiment is shown
in Fig. 1A, where picrotoxin inhibition was measured at
voltages from -70 to +50 mV in 20-mV steps. The
current-voltage relationships derived from this experiment are shown in
Fig. 1B. It is clear that there is no significant
voltage-dependence of picrotoxin inhibition over this voltage range.
Similar results were found in each of four cells using this voltage
clamp procedure and in six other cells using digitally-generated
voltage ramps from -90 to +90 mV (data not shown).
Figure 1:
Picrotoxin-induced
inhibition is not voltage-dependent. A, superimposed
voltage-clamp recordings from a cell held at membrane potentials of
between -70 and +50 mV at -20 mV intervals. In this
and all subsequent figures, the dashedline represents the zero current level, and downwarddeflections represent current flowing into the cell. For
all traces, the hollowbar represents the application
of 1 mM glycine, and the filledbar represents the addition of 30 µM picrotoxin. B, Current-voltage relationships for glycine-activated currents
(hollowcircles) and with added picrotoxin
(filledcircles) derived from the recordings
displayed in A.
In
GABARs, the rate of onset and washout of the
picrotoxin-induced inhibition is dramatically enhanced in the presence
of GABA
(27, 28, 29) , suggesting that its
binding site is exposed following the binding of GABA to the receptor.
The possible use dependence of picrotoxin on GlyRs was examined using
several strategies. When 100 µM picrotoxin was co-applied
with 1 mM glycine, a brief transient peak current was
elicited, which decayed rapidly (within 300 ms) to a constant
steady-state level, with no evidence for a slowly-developing inhibitory
component (Fig. 2A, filledcircle). A
1-s preexposure to picrotoxin abolished this spike, but it did not
affect the steady-state inhibition level (n = 5; data
not shown). When picrotoxin was subsequently removed in the maintained
presence of glycine, the rate of channel reactivation was significantly
slower (Fig. 2A, filledcircle) than
when glycine was applied in the absence of picrotoxin preexposure
(Fig. 2A, hollowcircle). To
facilitate this comparison, the activation segments of each trace are
normalized, superimposed, and expanded in Fig. 2C. In a
total of 5 cells, the time to half-maximal activation of glycine-gated
currents without picrotoxin preexposure was significantly increased
after a 1.5-s preexposure to picrotoxin (Fig. 2D).
Details of the statistical analysis used to assess significance are
given in the legend to Fig. 2. Thus, the reduced rate of current
activation following washout of picrotoxin may be used as an assay of
the dissociation of picrotoxin from its receptor.
Figure 3:
Repeated applications of 1 mM
glycine in the constant presence of 100 µM picrotoxin do
not lead to accumulated inhibition. A, the application of 100
µM picrotoxin is indicated by the filledbar, and the applications of 1 mM glycine are
indicated by the hollowbars and numbers.
B, the proportion of current remaining following glycine
application plotted as a ratio of the response to the first glycine
application. Data are averaged from four cells and fitted by linear
regression.
We examined the
picrotoxin-induced inhibition of glycine-gated currents in WT GlyRs to
determine whether or not it was a competitive antagonist. Picrotoxin
inhibitory dose-response curves were measured in the presence of both a
saturating concentration (1 mM) and an approximately
half-saturating concentration (30 µM) of glycine. An
example of an inhibitory dose-response for 30 µM
picrotoxin is shown in Fig. 4A. In
Fig. 4B, picrotoxin dose responses were averaged from
five cells in 30 µM glycine (squares) and for six
other cells in 1 mM glycine (circles).
Averaged results for the picrotoxin half-maximal inhibitory
concentration (IC) and Hill coefficients are presented in
, and -fold increases in IC
s are shown in
parentheses. The increased glycine concentration resulted in an
8.1-fold increase in the IC
for picrotoxin, clearly
revealing a strong competition between glycine and picrotoxin. To
determine whether the picrotoxin antagonism was completely or partially
competitive, the degree of inhibition induced by 10 µM
picrotoxin was measured at glycine concentrations of 10, 30, 100, and
1000 µM. As shown in Fig. 5A, at 10
µM glycine, picrotoxin inhibition was complete, and this
was progressively overcome as the glycine concentration was increased
to 1 mM. Averaged results from five cells, each recorded in
both 0 and 10 µM picrotoxin, are displayed in
Fig. 5B as circles andsquares,
respectively. In the absence of picrotoxin, the glycine half-maximal
activation concentration (EC
) was 25 ± 3
µM (±S.E., n = 5) with a Hill
coefficient of 1.8 ± 0.1, and in the same five cells in the
presence of 10 µM picrotoxin, the EC
increased approximately 4-fold to 95 ± 16, while the Hill
coefficient (1.6 ± 0.1) was not significantly changed. These
results indicate that picrotoxin acts purely as a competitive
antagonist of glycine in WT GlyRs.
Figure 4:
The efficacy of picrotoxin inhibition is
dependent on the glycine concentration. A, an example of a
picrotoxin dose response in 30 µM glycine. B, averaged dose responses of picrotoxin inhibiton from five cells in
30 µM glycine (squares) and six cells in 1
mM glycine (circles).
Figure 5:
Inhibition induced by 10 µM
picrotoxin in WT GlyRs is progressively overcome by increasing the
glycine concentration. A, a series of current traces from one
cell showing that the inhibition induced by 10 µM
picrotoxin is overcome by increasing the glycine concentration from 10
µM to 1 mM. Apparent concentration-dependent
differences in picrotoxin onset kinetics may be complicated by chloride
shift effects (Rajendra et al., 1995). B, averaged
glycine dose responses in 0 and 10 µM picrotoxin. All data
were recorded from the same five cells.
Picrotoxin is an equimolar
mixture of picrotoxinin and picrotin, of which only picrotoxinin is
active in antagonizing the GABAR
(19) . The relative
potencies of picrotoxinin and picrotin have not been previously tested
on the GlyR. As seen in Fig. 6A, both picrotin and
picrotoxinin were similar in their ability to antagonize the currents
activated by 1 mM glycine, but each was less efficacious than
picrotoxin. In Fig. 6B, averaged inhibitory dose
responses are displayed for both picrotin (uprighttriangles) and picrotoxinin (invertedtriangles), together with the results for picrotoxin
(hollowcircles) replotted from
Fig. 4B. The respective IC values and Hill
coefficients are shown in . There is clearly no
significant difference in relative efficacies of picrotin and
picrotoxinin in inhibiting the glycine-induced current (unpaired t test, p > 0.05). To reconstitute the effect of
picrotoxin from the sum of its constituents, the combined inhibitory
dose response of picrotin and picrotoxinin was calculated by
multiplying together the fractional currents remaining after exposure
to each constituent at each concentration. These points were then
fitted by the curve shown as a dashedline in
Fig. 6B. This curve had an IC
and Hill
coefficient as given in , which was not significantly
different from the averaged values for picrotoxin (unpaired t test, p > 0.05).
Figure 6:
Picrotin and picrotoxinin are equally
efficacious in inhibiting glycine-gated currents in WT GlyRs. A, series of current traces recorded from one cell sequentially
exposed to 30 µM concentrations of picrotoxin,
picrotoxinin, and picrotin (filledbars), all in the
presence of 1 mM glycine (hollowbars).
B, inhibitory dose responses of picrotin (uprighttriangles) and picrotoxinin (invertedtriangles) averaged from four and three cells,
respectively. The calculated combined effect of these inhibitors is
indicated by the dashedline. The picrotoxin
inhibition curve (hollowcircles), replotted from
Fig. 4, is included for comparison.
Action of Picrotoxin on R271L and R271Q GlyRs
When
tested on GlyRs expressing either of the two startle disease mutations,
Arg-271 Leu (R271L) or Arg-271
Gln (R271Q), picrotoxin
caused both a potentiation and an inhibition of glycine-gated currents
in the presence of a submaximal (5 mM) glycine concentration.
Examples of picrotoxin dose responses for both mutant receptors are
shown in Fig. 7A. At a picrotoxin concentration of 1
µM, only a potentiation was observed. At 3
µM, the potentiation was rendered transient by the
superposition of a slowly developing inhibition. At higher picrotoxin
concentrations (10 and 30 µM), only a
concentration-dependent inhibition was observed. Thus, in both mutated
GlyRs, picrotoxin appeared to exert two opposite functions, which
overlapped at 3 µM. The potentiation was not due to
picrotoxin acting as an agonist. As shown in Fig. 7B,
for both the R271L and R271Q GlyRs, application of 0.3 µM
picrotoxin alone was not sufficient to activate a current, although
when applied with 5 mM glycine it was able to strongly
potentiate the glycine-gated current. This was observed in each of five
cells expressing each mutant receptor. Accordingly, at low
concentrations, picrotoxin acts as an allosteric potentiator of
glycine-gated currents. This contrasts with the WT GlyR, where at a
half-maximal glycine concentration (30 µM) and a
subthreshold picrotoxin concentration (0.3 µM), no
potentiation was ever observed (Fig. 4).
Figure 7:
Effects of picrotoxin on R271L and R271Q
GlyRs. A, at a submaximal glycine concentration of 5
mM, picrotoxin induces an increase in the glycine-gated
current at 1 µM, an increase followed by a slow onset
inhibition at 3 µM, and an inhibition only at 10 and 30
µM picrotoxin. Similar results were obtained for both
R271L and R271Q mutant GlyRs (left and rightpanels, respectively). Numbers represent
picrotoxin concentrations in µM. Unless otherwise
indicated, the numbers to the left and right of the
verticalcalibrationbars apply to figures
to the left and right, respectively. B, a
concentration of 0.3 µM picrotoxin alone (filledbar) does not activate currents in either the R271L
(left panel) or R271Q (rightpanel) GlyRs,
but at the same concentration is able to strongly potentiate the
currents activated by 5 mM glycine (hollowbar) in both mutant GlyRs.
We sought to
establish whether the potentiating and inhibitory effects of picrotoxin
were mediated through binding sites with different characteristics. As
a first approach, we investigated whether both effects were competitive
with glycine. To isolate the potentiating from the inhibitory effects,
a picrotoxin concentration of 0.3 µM was used, as it is
below the threshold for inhibition. The potentiation, expressed as a
percentage of the maximum activable current, was measured at glycine
concentrations of 5, 20, and 100 mM. A concentration of 5
mM is approximately half-saturating, whereas a 100 mM
concentration is saturating. The EC for glycine activation
in the R271L GlyR is 6.7 mM and in the R271Q GlyR is 12
mM (Rajendra et al., 1994). As shown in
Fig. 8A, the picrotoxin-induced potentiation was
substantial at 5 mM, but in both mutants, GlyR was
progressively completely diminished by 100 mM glycine. The
percentage current increases averaged from three cells expressing the
R271L and R271Q GlyRs are displayed in Fig. 8B. These
results indicate that the picrotoxin-induced potentiation is the result
of a decrease in the glycine EC
, with no change to the
maximum peak current amplitude.
Figure 8:
Picrotoxin-induced potentiation is caused
by an increased glycine affinity of both R271L and R271Q GlyRs. A, examples of the effects produced by 0.3 µM picrotoxin
on currents elicited by 5, 20 and 100 mM glycine in both R271L
(left) and R271Q (right) GlyRs. B, averaged percentage
increases in the peak glycine-activated current at the glycine
concentrations of 5, 20, and 100 mM for both R271L
(left) and R271Q (right) GlyRs. Data were averaged
from three cells for each mutant receptor, and errorbars indicate S.E. At the subthreshold glycine concentration of 200
µM, the application of 0.3 µM picrotoxin had
no effect (n = 3).
To investigate the potentiation
further, the dose response of the picrotoxin-induced potentiation was
examined in both mutant GlyRs, using picrotoxin concentrations between
0.01 and 3 µM. A constant glycine concentration of 5
mM was used throughout. An example of a dose-response curve
for a R271Q GlyR is given in Fig. 9A. It displayed a
clear bell-shaped concentration dependence, and the rising and falling
phases are presented in separate panels for clarity. At 3
µM, the slow onset inhibitory effect can also be observed.
The averaged dose-responses measured in four cells expressing R271Q
GlyRs are shown in Fig. 9B (squares), where the
percentage current increase is displayed relative to the mean current
amplitude before picrotoxin application. From this figure, it can be
seen that the threshold for potentiation occurs at less than 0.01
µM picrotoxin, reaches a maximum near 0.3 µM
and is substantially diminished by 3 µM. The averaged
dose-response was also measured from 4 cells expressing the R271L GlyR
(Fig. 9B, circles). This displayed a similar
maximal potentiation but peaked at the lower picrotoxin concentration
of 0.1 µM.
Figure 9:
Picrotoxin-induced potentiation has a
bell-shaped dose-response curve. A, an example of a picrotoxin
potentiation dose response in R271Q GlyRs. Picrotoxin concentrations of
0.01-3 µM were applied in the presence of 5
mM glycine to a cell expressing R271Q GlyRs. Numbers represent
picrotoxin concentration in µm. All data are from the same cell.
The rising and falling phases of the bell are displayed in the upper
and lower panels, respectively. Note overlap between potentiation and
inhibition at 3 µM. B, Potentiation dose
responses averaged from four cells expressing the R271L GlyR
(circles) and from four cells expressing the R271Q GlyR
(squares). Errorbars represent
S.E.
The inhibitory effect of picrotoxin was also
examined for possible competition with glycine. Picrotoxin inhibitory
dose responses were measured using glycine concentrations of 5 and 100
mM, and picrotoxin concentrations of 3, 10, 30, and 100
µM. Sample traces displaying the picrotoxin-induced
inhibiton in both mutant GlyRs are shown for both 100 and 5 mM
glycine in Fig. 10A (upper and lowerpanels), respectively. Although the inhibition in
response to 30 and 10 µM picrotoxin appeared similar for
both 5 and 100 mM glycine in both mutant GlyRs, using 3
µM picrotoxin resulted in an inhibition at 100 mM
glycine, but a potentiation at 5 mM glycine. This is expected
due to the glycine-dependence of the potentiation (Fig. 8) and to
the overlap between potentiation and inhibition at 3 µM
picrotoxin (e.g.Fig. 7A and 9A).
Accordingly, at 5 mM glycine, longer (5 s) exposures to 3
µM picrotoxin were required for the picrotoxin-induced
inhibition to reach a steady state level (Fig. 7A).
Picrotoxin inhibitory dose responses were measured in five cells, each
expressing R271L and R271Q GlyRs, and averaged results are shown in
Fig. 10B for both 100 mM glycine
(circles) and 5 mM glycine (squares). The
respective picrotoxin ICs and Hill coefficients are given
in , and -fold increases between half-maximal and maximal
glycine concentrations are indicated by numbers in parentheses. Whereas
the WT GlyR showed an 8.1-fold increase in the IC
over
this range, there was no significant change (unpaired t test;
p > 0.05) in the corresponding values for either of the
mutant GlyRs. Hence, picrotoxin is converted from a competitive
antagonist of the WT GlyR to a noncompetitive antagonist of both the
R271L and R271Q GlyRs.
Figure 10:
Picrotoxin inhibition is noncompetitive
in both R271L and R271Q GlyRs. A, the leftpanel shows examples of picrotoxin-induced inhibition of currents
activated at both 100 mM (upperpanel) and 5
mM (lowerpanel), in the same cell
expressing R271L GlyRs. Note the transient potentiation induced by 3
µM picrotoxin in the lowerpanel. The
upper and lowerrightpanels show
examples of corresponding data recorded in a single cell expressing
R271Q GlyRs. B, the leftpanel shows
averaged picrotoxin inhibitory dose-responses from 5 cells in 100
mM glycine (circles) and eight cells in 5 mM
glycine (squares) in R271L mutant GlyRs. Errorbars are shown when larger than symbol size. The
rightpanel shows corresponding data for the R271Q
mutant GlyR. Data were averaged from five cells in 100 mM
glycine (circles) and seven cells in 5 mM glycine
(squares).
As described above (), both
picrotoxinin and picrotin were equally efficacious in antagonizing the
WT GlyR. In an attempt to pharmacologically differentiate between the
potentiating and inhibitory effects of picrotoxin in the R271L and
R271Q GlyRs, the efficacies of both picrotoxinin and picrotin were
evaluated on both response types. To examine potentiation in the
absence of inhibition, a 5 mM concentration of glycine was
used with picrotoxin, picrotoxinin, and picrotin at concentrations of
0.1 µM. To examine inhibition in the absence of
potentiation, a 100 mM glycine concentration was used with a
30 µM concentration of each ligand. The efficacies of the
three compounds in inhibiting the R271L and R271Q GlyRs were averaged
from three cells each, and the results are summarized in
I. In both mutant GlyRs there was no significant
difference in the inhibition produced by picrotin or picrotoxinin, but
as expected, the inhibition produced by picrotoxin was significantly
stronger (paired t test; p < 0.05). Picrotoxinin
and picrotin were also equally efficacious in potentiating
glycine-activated currents. As shown in I, in four cells
expressing the R271L GlyR and in five cells expressing the R271Q GlyR,
there was no significant difference (paired t test) in the
amount of potentiation induced by either component.
Picrotoxin Inhibition of WT GlyR: Comparison with
Effects on GABA
Although picrotoxin is
known to act as a potent antagonist of homomeric
GlyRs
(20, 30) , the mechanism of its action has not been
previously analyzed in detail. In this study, we found that the nature
of picrotoxin antagonism of the GlyR differs fundamentally from that
observed at the GABARR. It has generally been found that
picrotoxin inhibition of the GABAR is independent of
voltage
(27, 29, 21, 32) , although at
least one study
(33) presented evidence for some voltage
sensitivity. In GlyRs, we found that, as expected for an uncharged
molecule, picrotoxin did not change the whole cell glycine
current-voltage relationship (Fig. 1B). Studies of
GABA-mediated responses have found that the time for development of
picrotoxin inhibition is reduced from 10 to 20 min in the absence of
agonist, to around 10 to 20 s in the presence of
agonist
(27, 28, 29) . Thus, the picrotoxin
binding site is exposed following GABA-induced activation of the
channel. In Fig. 2, we demonstrated that picrotoxin binding had
reached a steady state at an equivalent rate whether in the presence or
absence of glycine. Furthermore, in Fig. 3it can be seen that
repeated glycine applications did not enhance the picrotoxin-induced
inhibition. Thus, contrary to the GABAR, there was no
detectable glycine dependence of picrotoxin efficacy.
R
(19) , but the relative potencies of these
compounds in antagonizing the GlyR has not previously been examined. To
our surprise, picrotin and picrotoxinin were equally efficacious in
antagonizing WT GlyRs (Fig. 7). This was not due to contamination
of the picrotin sample by picrotoxinin because each compound was
approximately half as effective as picrotoxin (Fig. 7B).
Based on the lack of discrimination between picrotoxinin and picrotin,
GlyRs appear to express a novel picrotoxin binding site. Since the
molecules differ only in the structure of the terminal isoprenyl group
(which in the case of picrotin is hydrated to remove the double bond),
the molecular orientation required for binding at the GlyR picrotoxin
site must be different from that required for activation of the
GABAR picrotoxin site.
Allosteric Inhibition by Picrotoxin in the WT
GlyR
The M2 region of the GlyR subunit has a low degree of
homology with those of known GlyR subunits
(41) . It was
recently demonstrated that / GlyR heteromers were strongly
resistant to picrotoxinin antagonism and that mutating the divergent M2
residues of the subunit back toward those of the subunit
restored picrotoxinin sensitivity
(20) . Since residues within
the corresponding region of the nAChR control ion permeation and
blocker binding
(8) , it was concluded that picrotoxin acted by
blocking the channel. However, in this report we have demonstrated that
picrotoxin is a purely competitive antagonist, which is difficult to
explain in terms of channel block
(8, 21) . The lack of
use dependence (Fig. 2) also supports this contention, since
channels must normally open before a blocker can bind to the central
region of the pore. Our results imply that, like all other known
competitive antagonists of ligand-gated ion channels
(8) ,
picrotoxin binds to a site in the extracellular domain. Its effects may
be exerted in one of two ways. Firstly, like strychnine, its binding
site may overlap that of glycine, resulting in antagonism by steric
hindrance
(16, 17) . In the case of picrotoxin, this
appears unlikely because radioligand binding experiments in our
laboratory have indicated that picrotoxin displaces bound
[
H]strychnine and displaces glycine displacement
of bound [H]strychnine with extremely low
(millimolar) affinities.()
The second possibility
is that like many competitive antagonists of the nAChR
(18) ,
picrotoxin may be allosterically coupled to a residue controlling ion
channel desensitization. This possibility is much more likely because,
as discussed in the next section, mutations to the Arg-271 transduction
site caused dramatic alterations in the picrotoxin transduction
mechanism, and the steric hindrance ability should not be affected by
mutations to a transduction residue.
R
channel blocker on the basis of its use dependence and noncompetitive
antagonism (e.g. 28), recent evidence from single channel
studies
(29, 43) , studies using synthetic picrotoxin
analogues
(31, 44) and picrotoxin-resistant
mutants
(45) , have suggested that picrotoxin may also be an
allosteric inhibitor of the GABAR.
Altered Picrotoxin Transduction in Mutated
GlyRs
In this report, we have demonstrated that in both R271L
and R271Q GlyRs, picrotoxin exerts both an allosteric potentiating
effect and a noncompetitive inhibitory effect. These results provide
evidence not only for the existence of two distinct functions but also
for two distinct binding sites. Although it is premature to propose a
model to explain these results, it is possible to make some inferences
about the role of Arg-271 in the coupling of the picrotoxin binding
sites to functional domains of the GlyR. The most conservative
interpretation is that the mutations have modified both the coupling
from an existing picrotoxin binding site and have either created, or
functionally coupled, a new binding site. It is much less plausible to
speculate that a single point mutation could abolish one and create two
new picrotoxin binding sites with different functional coupling
mechanisms.
Rs are
thought to contain both competitive and noncompetitive picrotoxin
binding sites
(31) . In addition, one picrotoxin analogue,
-ethyl--methyl -butryrolactone, also potentiates GABA
responses at low (0.5 µM) concentrations
(44) .
Thus, picrotoxin action on mutated GlyRs appears to have some
similarities to its effects on native GABA
Rs.
Possible Therapeutic Role for Picrotin-induced
Potentiation in Human Startle Disease
The R271L and R271Q GlyR
mutations have been shown to underlie human hereditary hyperekplexia,
or startle disease
(11) , by inducing both a decrease in current
amplitude and a loss of sensitivity of GlyRs to activation by
glycine
(13, 14) . In mutated GlyRs with the putative
in vivo subunit stoichiometry, glycinergic currents are about
25% of their magnitude in normal GlyRs
(14) . The demonstration
in this report that the startle disease mutations result in the
creation of a high affinity site for allosteric potentiation has
possible therapeutic implications. At low concentrations, either
picrotoxinin or picrotin can dramatically potentiate glycinergic
currents, thus at least partially overcoming the deleterious effect of
the mutations. Picrotin would be the more suitable therapeutic
candidate, because unlike picrotoxinin, it does not block GABAergic
neurotransmission.
Table:
Picrotoxin inhibition of channel activation of
wild-type and mutated GlyRs
), the Hill coefficients,
their standard errors, and the number of determinations (n)
are shown for both WT and mutated GlyRs. The two glycine concentrations
used in each case represent approximately half-saturating and
saturating concentrations, respectively. -Fold increases of saturating
with respect to half-saturating values are shown in parentheses.
Table:
Relative potencies of picrotoxin, picrotoxinin,
and picrotin in inhibiting the WT GlyR
Table:
Relative efficacies of picrotoxinin and
picrotin in inducing potentiation and inhibition in R271L and R271Q
GlyRs
-aminobutyric acid; nAchR, nicotinic acetylcholine receptor;
GABA
R, GABA type A receptor; WT, wild-type.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  R. E. Wiltfong and M. Jansen Probing Protein Packing Surrounding the Residues in and Flanking the Nicotinic Acetylcholine Receptor M2M3 Loop J. Neurosci., February 11, 2009; 29(6): 1626 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.-S. Wang, R. Buckinx, H. Lecorronc, J.-M. Mangin, J.-M. Rigo, and P. Legendre Mechanisms for Picrotoxinin and Picrotin Blocks of {alpha}2 Homomeric Glycine Receptors J. Biol. Chem., June 1, 2007; 282(22): 16016 - 16035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sedelnikova, B. E. Erkkila, H. Harris, S. O. Zakharkin, and D. S. Weiss Stoichiometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor J. Physiol., December 1, 2006; 577(2): 569 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-S. Wang, J.-M. Mangin, G. Moonen, J.-M. Rigo, and P. Legendre Mechanisms for Picrotoxin Block of {alpha}2 Homomeric Glycine Receptors J. Biol. Chem., February 17, 2006; 281(7): 3841 - 3855. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Hawthorne and J. W. Lynch A Picrotoxin-specific Conformational Change in the Glycine Receptor M2-M3 Loop J. Biol. Chem., October 28, 2005; 280(43): 35836 - 35843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Mangin, L. Nguyen, C. Gougnard, G. Hans, B. Rogister, S. Belachew, G. Moonen, P. Legendre, and J.-M. Rigo Developmental Regulation of {beta}-Carboline-Induced Inhibition of Glycine-Evoked Responses Depends on Glycine Receptor {beta} Subunit Expression Mol. Pharmacol., May 1, 2005; 67(5): 1783 - 1796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Qian, Y. Pan, Y. Zhu, and P. Khalili Picrotoxin Accelerates Relaxation of GABAC Receptors Mol. Pharmacol., February 1, 2005; 67(2): 470 - 479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Lynch Molecular Structure and Function of the Glycine Receptor Chloride Channel Physiol Rev, October 1, 2004; 84(4): 1051 - 1095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. L. Absalom, T. M. Lewis, and P. R. Schofield Mechanisms of channel gating of the ligand-gated ion channel superfamily inferred from protein structure Exp Physiol, March 1, 2004; 89(2): 145 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Thio, A. Shanmugam, K. Isenberg, and K. Yamada Benzodiazepines Block {alpha}2-Containing Inhibitory Glycine Receptors in Embryonic Mouse Hippocampal Neurons J Neurophysiol, July 1, 2003; 90(1): 89 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M Lewis, P. R Schofield, and A. M L McClellan Kinetic determinants of agonist action at the recombinant human glycine receptor J. Physiol., June 1, 2003; 549(2): 361 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J M Mangin, A Guyon, D Eugene, D Paupardin-Tritsch, and P Legendre Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra J. Physiol., August 1, 2002; 542(3): 685 - 697. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Dibas, E. B. Gonzales, P. Das, C. L. Bell-Horner, and G. H. Dillon Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine alpha 1 Receptors J. Biol. Chem., March 8, 2002; 277(11): 9112 - 9117. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-Q. Huang, C. L. Bell-Horner, M. I. Dibas, D. F. Covey, J. A. Drewe, and G. H. Dillon Pentylenetetrazole-Induced Inhibition of Recombinant gamma -Aminobutyric Acid Type A (GABAA) Receptors: Mechanism and Site of Action J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 986 - 995. [Abstract] [Full Text]
|
|
|
|
|
|
J. W. Lynch, N.-L. R. Han, J. Haddrill, K. D. Pierce, and P. R. Schofield The Surface Accessibility of the Glycine Receptor M2-M3 Loop Is Increased in the Channel Open State J. Neurosci., April 15, 2001; 21(8): 2589 - 2599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J Smith, S. Owens, and I. D Forsythe Characterisation of inhibitory and excitatory postsynaptic currents of the rat medial superior olive J. Physiol., December 15, 2000; 529(3): 681 - 698. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Steinbach, J. Bracamontes, L. Yu, P. Zhang, and D. F. Covey Subunit-Specific Action of an Anticonvulsant Thiobutyrolactone on Recombinant Glycine Receptors Involves a Residue in the M2 Membrane-Spanning Region Mol. Pharmacol., July 1, 2000; 58(1): 11 - 17. [Abstract] [Full Text]
|
|
|
|
|
|
S. Levi, D. Chesnoy-Marchais, W. Sieghart, and A. Triller Synaptic Control of Glycine and GABAA Receptors and Gephyrin Expression in Cultured Motoneurons J. Neurosci., September 1, 1999; 19(17): 7434 - 7449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Moorhouse, P. Jacques, P. H. Barry, and P. R. Schofield The Startle Disease Mutation Q266H, in the Second Transmembrane Domain of the Human Glycine Receptor, Impairs Channel Gating Mol. Pharmacol., February 1, 1999; 55(2): 386 - 395. [Abstract] [Full Text]
|
|
|
|
|
|
A. Stumpner Picrotoxin Eliminates Frequency Selectivity of an Auditory Interneuron in a Bushcricket J Neurophysiol, May 1, 1998; 79(5): 2408 - 2415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T M Lewis, L G Sivilotti, D Colquhoun, R M Gardiner, R Schoepfer, and M Rees Properties of human glycine receptors containing the hyperekplexia mutation {alpha}1(K276E), expressed in Xenopus oocytes J. Physiol., February 15, 1998; 507(1): 25 - 40. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Legendre Pharmacological Evidence for Two Types of Postsynaptic Glycinergic Receptors on the Mauthner Cell of 52-h-Old Zebrafish Larvae J Neurophysiol, May 1, 1997; 77(5): 2400 - 2415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ueno, J. Bracamontes, C. Zorumski, D. S. Weiss, and J. H. Steinbach Bicuculline and Gabazine Are Allosteric Inhibitors of Channel Opening of the GABAA Receptor J. Neurosci., January 15, 1997; 17(2): 625 - 634. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.R. Schofield, J.W. Lynch, S. Rajendra, K.D. Pierce, C.A. Handford, and P.H. Barry Molecular and Genetic Insights into Ligand Binding and Signal Transduction at the Inhibitory Glycine Receptor Cold Spring Harb Symp Quant Biol, January 1, 1996; 61(0): 333 - 342. [Abstract] [PDF]
|
|
|
|
|
|
Q. Shan, J. L. Haddrill, and J. W. Lynch Ivermectin, an Unconventional Agonist of the Glycine Receptor Chloride Channel J. Biol. Chem., April 13, 2001; 276(16): 12556 - 12564. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |