©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutations Affecting the Glycine Receptor Agonist Transduction Mechanism Convert the Competitive Antagonist, Picrotoxin, into an Allosteric Potentiator (*)

Joseph W. Lynch (1)(§), Sundran Rajendra (2), Peter H. Barry (2), Peter R. Schofield (1)

From the (1) Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, New South Wales, 2010, Australia and the (2) School of Physiology and Pharmacology, University of New South Wales, Sydney, New South Wales, 2052, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Contrary to its effects on the -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.


INTRODUCTION

Glycine receptors (GlyRs)() 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.

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 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.

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 (GABARs) 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.


EXPERIMENTAL PROCEDURES

Mutation and Expression of Human GlyR Subunit cDNA

Mutations in the cDNA encoding the human GlyR 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.

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.


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.




RESULTS

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.

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.


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 ICs 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.


DISCUSSION

Picrotoxin Inhibition of WT GlyR: Comparison with Effects on GABAR

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 GABAR. 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.

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 GABAR (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.

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 GABAR 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.

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, GABARs 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 GABARs.

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.

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 values of the picrotoxin half-inhibitory concentration (EC), 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

All experiments were performed using a glycine concentration of 1 mM.


  
Table: Relative efficacies of picrotoxinin and picrotin in inducing potentiation and inhibition in R271L and R271Q GlyRs

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.



FOOTNOTES

*
This work was supported by the Australian National Health and Medical Research Council and the Vincent Fairfax Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 61-2-361-2050; Fax: 61-2-332-4876.

The abbreviations used are: GlyR, glycine receptor; GABA, -aminobutyric acid; nAchR, nicotinic acetylcholine receptor; GABAR, GABA type A receptor; WT, wild-type.

C. A. Handford and P. R. Schofield, unpublished observations.


ACKNOWLEDGEMENTS

We thank Vikki Falls, Cheryl Handford and Kerrie Pierce for excellent technical assistance.


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