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J. Biol. Chem., Vol. 281, Issue 7, 3841-3855, February 17, 2006

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Mechanisms for Picrotoxin Block of ₂ Homomeric Glycine Receptors^*

Dian-Shi Wang¹, Jean-Marie Mangin^¶, Gustave Moonen^¶, Jean-Michel Rigo^¶||, and Pascal Legendre²

From the Unité Mixte de Recherche, CNRS 7102, Neurobiologie des Processus Adaptatifs, Université Pierre et Marie Curie, 9 Quai St. Bernard, 75252, Paris cedex 05, France, the ^¶FacultédeMédecine de Liège, Centre de Recherche en Neurobiologie Cellulaire et Moléculaire, 17 place Delcour, B-4020 Liège 2, Belgium, the ^||Biomed Research Center, Universiteit Hasselt, Agoralaan, B-3590 Diepenbeek, Belgium, and the Department of Anatomy, Fourth Military Medical University, Xi'an 710032, China

Received for publication, October 11, 2005 , and in revised form, December 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal -aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of ₂ homomeric GlyR is still unclear compared with that of ₁ homomeric GlyR, GABA_A, and GABA_C receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by ₂ homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the ₂ homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the ₂ homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric ₂ GlyR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Glycine and GABA³ are the main inhibitory neurotransmitters in the central nervous system. The glycine receptor (GlyR) is a pentameric transmembrane protein complex, which forms an anion-selective channel (1). Five different subunits have hitherto been cloned in mammals, one subunit and four subunits (₁–₄), which are associated with two different ways of forming functional receptors: the homomeric configuration composed of five subunits (1) and the heteromeric configuration comprising two subunits and three subunits (2). In the adult brain, GlyR is primarily involved in fast inhibitory synaptic transmission in the brainstem and spinal cord.

It is now well established that picrotoxin (PTX), a plant alkaloid, which was first used to discriminate GABAergic from glycinergic currents, can also strongly inhibit the homomeric GlyR subtypes, whereas the / heteromeric GlyR subtype is much less sensitive to PTX (3). Although the action of PTX has been extensively studied both on GABA_A and GABA_C receptors and on GlyRs (4), the one or more binding sites of this compound and its inhibitory mechanism are still under debate. There are lines of evidence indicating that PTX binding and/or inhibitory mechanisms are related to amino acid residues in the transmembrane domain TM2 forming the pore of the ionic channel (3, 5–13). A series of studies on the GABA_AR, GABA_CR, invertebrate glutamate receptor Cl^– channel, and GlyRs has established that a ring of 6'-threonines within the pore is invariably required for PTX sensitivity (4). Recently, it has been accurately demonstrated that PTX is likely to bind in the channel pore of the homomeric ₁ GlyR (14). Although PTX could be trapped when the GlyR channel closed in the ₁ subunit R271C mutation, this was not the case for wild-type GlyR (14), and it is unlikely that PTX can act as an open channel blocker on this GlyR subtype. In fact, the inhibitory mechanism of PTX can differ between anionic receptor-channel family subtypes, and it ranges from open channel blocker to allosteric competitive antagonist (4). Although in some preparations PTX inhibition of GABA_AR appeared to be use-dependent and noncompetitive (15), suggesting an open channel block mechanism for PTX inhibition, analysis of PTX-evoked inhibition of the single-channel activity of GABA_AR recorded from rat sympathetic neurons indicates that PTX inhibition is not use-dependent (16). In this study, the authors proposed that PTX preferentially binds to the agonist-bound conformation of the receptor and stabilized the channel in the closed state (16). This was also demonstrated with GABA_C receptors from isolated retinal bipolar cells and from oocytes expressing the GABAR subunit (17). A competitive inhibitory component of PTX inhibition has been described for homomeric ₁ GlyR. Its potency decreased as agonist concentration increased (18). But PTX is unlikely to modify glycine binding, and it was proposed that PTX rather acts as an allosteric inhibitor by altering the coupling between agonist binding and channel gating (18). Although extensive efforts have been made to determine the molecular mechanism of PTX inhibition of GlyRs, paradoxically little work has been done on the single-channel effects of PTX on GlyR kinetics. It has only been shown that at low concentrations (1–30 µM) PTX decreased the probability of predominantly high conductance with homomeric ₁ GlyRs. In contrast, at higher concentrations PTX induced flickering closures in both heteromeric ₁/ GlyRs and homomeric ₁ GlyRs (19, 20).

The ₂ homomeric GlyR subunit has been identified as an embryonic receptor form (21, 22) and plays an important role during synaptogenesis and cell differentiation. It is more adapted to sustained and slow paracrine neurotransmitter release (23) as observed in the fetal brain (24). It has recently been shown that PTX is as potent on homomeric ₂ GlyRs (25) as on homomeric ₁ GlyRs (3, 18, 19). Furthermore, ₂ homomeric GlyR has slow kinetic properties (23) and opens mainly with a single large conductance state (100–120 pS), which makes this receptor a good model for analyzing the effect of PTX on GlyR kinetics.

Therefore, we investigated here the mechanism of inhibition of PTX on the activation and deactivation kinetics of the homomeric ₂ GlyR expressed in Chinese hamster ovary (CHO) cells as a model system, by means of outside-out patch-clamp recordings using an ultra-fast flow application system. We showed that PTX inhibited ₂ homomeric GlyRs in a concentration-dependent and voltage-independent manner, and that PTX could bind to the receptor in both the open channel conformation and the fully liganded closed state. We also demonstrated that PTX could be trapped at its binding site when the channel closed during glycine dissociation. This complex mechanism can be predicted by a simple kinetic model in which glycine can dissociate while PTX remains bound.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture—Chinese hamster ovary cells (CHO-K1, ATCC number CCL61) were maintained in a 95% air-5% CO₂ humidified incubator at 35 °C in Dulbecco's modified Eagle's medium supplemented with 0.11 g/liter sodium pyruvate, 6 g/liter D-glucose, 10% (v/v) heat-inactivated fetal bovine serum (all from Invitrogen). Cells were passaged every 5–6 days (up to 20 times). For electrophysiological recordings, cells were seeded onto glass coverslips coated with poly-L-ornithine (0.1 mg/ml). Glycine receptor ₂ subunit cloning and transfection were performed as previously described (23).

Outside-out Recordings—Outside-out recordings (26) were done under direct visualization on ₂ GlyR-transfected CHO cells with the use of Normaski optics (x40, immersion lens, Nikon Optiphot). Cells were continuously perfused at room temperature (20–22 °C) with oxygenated bathing solution (2 ml/min) containing (in mM): NaCl 147, KCl 2.4, CaCl₂ 2, MgCl₂ 2, HEPES 10, glucose 10 (pH 7.2, osmolarity of 320 mosM). Patch-clamp electrodes (5–10 M) were pulled from thick-wall borosilicate glass with an outer diameter of 1.5 mm and inner diameter of 0.86 mm (Harvard Apparatus, Kent, UK) in multiple steps using a Brown-Flaming puller (Sutter Instrument Co., Novato, CA). They were fire-polished and filled with (in mM): CsCl 130, MgCl₂ 4, Na₂ATP 4, EGTA 10, HEPES 10 (pH 7.2, osmolarity of 290 mosM). Currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) and stored using a digital recorder (PCM-R300, Sony, Tokyo, Japan). Recordings were filtered at 10 kHz using an eight-pole Bessel filter (Frequency Devices, Haverhill, MA), sampled at 50 kHz and stored on a PC computer using pClamp software 6.03 (Axon Instruments). The membrane potential was held at –50 mV throughout the experiment, except when examining the I-V relationship. Patch currents represent the average of 10 or more trials as specified in the figure legends or the text unless otherwise noted.

Drug Delivery—Outside-out single-channel currents were evoked using a fast-flow operating system (27, 28). Control and drug solution were gravity-fed into two channels of a thin-wall glass theta tube (2-mm outer diameter, Hilgenberg, Malsfeld, Germany) pulled and broken to obtain a 200-µm tip diameter. The outside-out patch was positioned (45° angle) 100 µm away from the theta tubing, to be close to the interface formed between the flowing control and drug solutions. One lumen of the theta tube was connected to reservoirs filled with solutions containing glycine and/or PTX. The solution exchange was performed by rapidly moving the solution interface across the tip of the patch pipette, using a piezoelectric translator (model P245.30, Physik Instrument, Waldbronn, Germany). Concentration steps of glycine lasting 1–1000 ms were applied with an interval of 10 s. Exchange time of 10–90% (<100 µs) was determined before each set of experiments by monitoring the change in the liquid junction potential evoked by the application of a 10%-diluted control solution to the open tip of the patch pipette (28). For the experiments requiring fast solution exchange between three different conditions (see Fig. 8), we used a homemade multibarreled application system composed of three horizontally aligned quartz tubes (inside diameter, 0.25 mm; outside diameter, 0.35 mm; Polymicro Technologies). Solution exchange was achieved by lateral movements, using a SF-77B fast-step perfusion system (Warner Instruments, Hamdell, CT). The complete solution change was achieved in 200–300 µs. Glycine and PTX were from Sigma. Stock solution of PTX was prepared in dimethyl sulfoxide and then diluted to an appropriate concentration with the above extracellular solution just before use. The final concentration (v/v) of dimethyl sulfoxide was not higher than 0.3%, which had no effect on ₂ homomeric GlyRs as verified by control experiments (data not shown).

Data Analysis—Outside-out currents were analyzed off-line on a G4 Macintosh using Axograph 4.9 software (Axon Instruments). The concentration-inhibition curve of PTX was fitted using the Hill equation (Equation 1),

(Eq. 1)

where I is the response in the presence of PTX, I_Con is the control response (i.e. the glycine response in the absence of PTX), IC₅₀ is the PTX concentration at which half of the glycine response is blocked, and n_H is the Hill coefficient. For each concentration tested, the amplitude of the current, I, was measured at the steady-state level. The activation time constants of glycine-evoked currents in the presence and absence of PTX were estimated by fitting the onset of the responses with a sum of exponential curves using Axograph 4.9 software. Decay time constants were obtained by fitting the first 750 ms of the decay phase with a sum of exponential curves using Axograph 4.9 software (Axon Instruments). The presence of one or more exponential components was tested by comparing the sum of squared errors of the fits (28, 29).

For single-channel analysis, patches with one channel were included only if channel activity was stable over sweeps. First latencies, open and closed time durations were measured manually using Axograph 4.9 software. First latency distributions were created using standard histogram techniques (30). For display purposes, open and closed time histograms show the distributions as log intervals with the ordinate on a square root scale. These distributions were fitted with the sum of several exponential curves. The fit was optimized with the least square method (31). The number of exponential components was determined by comparing the sum of squared errors of the fits.

Kinetic Modeling Programs—To obtain a kinetic model for PTX effects on GlyR behavior, glycine-evoked currents in the absence and presence of PTX were analyzed off-line, and the inhibitory effect of PTX on GlyR kinetics was mathematically modeled using the chemical kinetic modeling programs included in the Axograph 4.9 software package (Axon Instruments). This program first calculated the change in the number of channels in each given state for given rate constants, and then systematically varied the rate constants to give the minimum sum of squared errors (SSEs) between the experimental data and a given model transient (29). Outside-out responses from 12 patches evoked by the application of glycine in the absence or presence of PTX were used for kinetic modeling analysis. Models were compared using the resulting SSE values of the fit.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Concentration-dependent inhibition of 2 homomeric GlyR by PTX. A, outside-out patch clamp recordings showing inhibition of 300 µM and 30 mM glycine-activated currents evoked by the indicated concentrations of co-application of PTX in CHO cells transfected with the 2 GlyR subunit. Each trace represents the average of 15–30 responses. Note that when the PTX concentration was >1 µM the small transient rebound current was always induced during the withdrawal of the two drugs. The left and right traces were obtained from two different patches. The thick line represents the application of drugs. B, PTX inhibition curves for 300 µM (•) and 30 mM () glycine-evoked responses. Currents were normalized to the responses in the absence of PTX. Each point is the average of values from 5–12 cells. In most instances multiple concentrations (three) of PTX were applied to the same cell. Data were fitted with the Hill equation (see "Materials and Methods") giving an IC50 of 2.7 ± 0.2 µM and a Hill coefficient of 0.8 ± 0.04 for 300 µM glycine, and an IC50 of 6.4 ± 0.6 µM and a Hill coefficient of 0.8 ± 0.05 for 30 mM glycine.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Concentration-dependent Inhibition of ₂ Homomeric GlyR by PTX— We first analyzed the ability of PTX to inhibit GlyR activity in terms of the outside-out current evoked by glycine applications to patches containing recombinant ₂ homomeric GlyR from CHO cells stably expressing the ₂ GlyR subunit. Fig. 1A illustrates the inhibitory effect of different concentrations of PTX on inward currents (V_H = –50 mV) evoked by 300 µM glycine (left traces, near the EC₅₀ for glycine-evoked response, see Ref. 23) and 30 mM glycine (right traces). In these experiments glycine was co-applied with PTX at concentrations indicated by the number below each trace. The co-application of PTX and glycine caused a concentration-dependent reduction of the current amplitude. The effect of PTX was reversible after washout. Interestingly, after co-application of 10 µM PTX and 300 µM glycine, when the two compounds were simultaneously withdrawn, a small transient membrane current was observed before current relaxation (Figs. 1A and 2A). Fig. 1B shows the concentration-response curves for PTX co-applied with two different concentrations of glycine obtained in twelve outside-out experiments. Concentration-response curves were fitted with the Hill equation (see "Materials and Methods") and in the presence of 300 µM glycine gave an IC₅₀ (half-maximum inhibition) and a Hill coefficient of 2.7 ± 0.2 µM and 0.8 ± 0.04, respectively. When glycine concentration was increased to 30 mM, the concentration-response curve was shifted to the right and the IC₅₀ value increased to 6.4 µM. The Hill coefficient value was not modified (0.8 in the presence of 30 mM glycine). This glycine concentration-dependent shift in PTX IC₅₀ was previously described for ₁ homomeric GlyRs and was thought to reflect a competitive inhibitory mechanism (18). However, it should be noted that PTX is unlikely to be a true competitive antagonist, because increasing the glycine concentration from 30 to 100 mM only slightly reduced the inhibitory effect of 10 µM PTX. The percentage inhibition of PTX-evoked outside-out current was 57 ± 2% (n = 12) and 50 ± 3% (n = 8) in the presence of 30 mM glycine and of 100 mM glycine, respectively.

PTX-induced Inhibition Is Not Voltage-dependent—The small rebound current observed during GlyR deactivation immediately after the termination of co-application of 10 µM PTX and glycine (Figs. 1A and 2A) could reflect the recovery from PTX open channel block as described for the open-channel block effect of acetylcholine on nicotinic receptors (32, 33). If PTX acts as a classic fast open channel blocker, the inhibitory effect of this alkaloid must be voltage-dependent. To test this hypothesis, the voltage dependence of PTX-induced glycine current inhibition was examined. Typical examples of 300 µM glycine-evoked currents (1-s application step) at V_H of +50 and –50 mV with and without co-application of 10 µM PTX are shown in Fig. 2A. In this example PTX inhibited glycine-evoked currents both at positive and at negative holding potentials. Voltage dependence of PTX inhibition on glycine-evoked currents was analyzed by constructing I-V curves from 300 µM glycine-evoked currents in the absence and presence of 10 µM PTX at holding potentials ranging from –70 mV to +70 mV (Fig. 2B). As shown in Fig. 2B, the steady-state current of the responses evoked in the absence or presence of PTX varied linearly at negative potentials and rectified at positive potentials. Adding PTX to glycine solution did not significantly change the reversal potential (V_r) of the glycine-evoked currents (unpaired t test, p > 0.1). V_r was 3 mV with glycine and 4 mV with glycine plus PTX. The voltage-independent nature of PTX block was revealed by plotting the percentage of block evoked by co-application of 10 µM PTX and 300 µM glycine at holding potentials ranging from +70 mV to –70 mV (Fig. 2C). Over this potential range PTX reduced the amplitude of glycine currents to the same extent (70%), indicating that PTX might not bind to a site within the membrane field of ₂ homomeric GlyRs. Nevertheless, PTX is weakly charged at neutral pH and the lack of a voltage-dependent block does not exclude the possibility that PTX can bind within the channel pore as recently suggested (14).

PTX Accelerates the Deactivation Kinetics of Glycine-evoked Current— A previous study has shown that PTX applied immediately after GABA accelerates the deactivation kinetics of GABA_CR (17). To determine if PTX has any effect on GlyR deactivation kinetics, we first compared the deactivation phase of the outside-out currents evoked by co-application of glycine and PTX or in the continuous presence of PTX before, during, and after glycine application (Fig. 3A). When compared with responses evoked by simultaneous application of 300 µM glycine and 10 µM PTX, the continuous presence of PTX dramatically accelerated the deactivation time course of the responses. In control conditions and after co-application of glycine and PTX the deactivation time constant (_decay) was 135 ± 9 ms (n = 13) and 133 ± 7 ms (n = 13), respectively. During the continuous presence of 10 µM PTX, the deactivation time constant was significantly decreased to 30 ± 3 ms (n = 13) (paired t test p < 0.01). Continuous application of PTX also accelerated the deactivation phase of responses evoked by a short (1 ms) step of a saturating concentration of glycine (30 mM, Fig. 3B1). As the PTX concentration increased, the deactivation time constants decreased in a concentration-dependent manner (Fig. 3B2). The decay phase of outside-out currents evoked by 1-ms application of 30 mM glycine was well fitted with a single exponential function, giving a _decay of 117 ± 12 ms (n = 10). In the presence of continuous PTX, _decay significantly decreased to 67 ± 2 ms for 1 µM PTX (n = 7), 45 ± 3 ms for 3 µM PTX (n = 10), 31 ± 4 ms for 10 µM PTX (n = 6), and 15 ± 2 ms for 30 µM PTX (n = 5), respectively (ANOVA, p < 0.01).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Voltage-independent inhibition of glycine response by PTX. A, responses to 300 µM glycine (control) and to co-application of 300 µM glycine and 10 µM PTX (glycine plus 10 µM PTX) at VH of +50 and –50 mV. Note that the rebound currents at both +50 and –50 mV are similar. Each trace represents the average of 10–12 trials. The thick line represents the application of 300 µM glycine. B, the current-voltage relationships of glycine responses induced by 300 µM glycine in the absence (•) and presence () of simultaneous application of 10 µM PTX. Note that the inhibitory effect of PTX is similar at all VH values. The reversal potentials of glycine-activated currents are 2.9 mV for glycine and 4.2 mV for glycine plus PTX. Currents were normalized to the response induced by 300 µM glycine alone at a VH of –50 mV (*). Each point is the mean of values from 5–10 cells. C, plot of the percentage of block by co-application of 300 µM glycine and 10 µM PTX as a function of the holding potentials. Data were averaged from 5–10 cells and fitted by linear regression.

To determine the microscopic determinants of the decrease in the decay time constant, we have analyzed the open time and closed time distributions in single receptor bursts of openings in response to short (1 ms) concentration pulses of glycine near GlyR saturation (30 mM) in the absence and presence of PTX. To perform this analysis, patches with a single functional GlyR were selected (i.e. patches that did not display superimposed openings in response to a saturating concentration of agonist; see Ref. 23). Single openings and closures were manually detected and measured using a filter cut-off frequency of 5 kHz. In control conditions, GlyR opens in bursts of long openings interrupted by very short closures (Fig. 4A). In the presence of continuous 10 µM PTX, the single opening duration appeared to be shortened (Fig. 4B). Opening and closing time constants were estimated by pooling measurements made on these single-channel responses obtained from 7–9 patches (23). The open time histograms were best fitted by single exponential curves both in control conditions and in the presence of continuous PTX (Fig. 4, C and D). In control conditions, the mean open time was 48.4 ms, which is consistent with the value we obtained previously (23). The mean open time was decreased to 6.1 ms in the presence of 10 µM PTX. In control conditions, the closed time distribution was best fitted by a single exponential curve with a closed time constant _c = 0.27 ms, as previously described for homomeric ₂ GlyRs (23). In the presence of PTX (10 µM) the closed time distribution was best fitted by the sum of two exponential curves giving a _c1 = 0.23 ms, which is very similar to the closed time constant in the control conditions. A second closed time was detected in the presence of PTX with a time constant _c2 = 5.76 ms (Fig. 4F). This longer closed time is likely to reflect an additional recovery pathway from PTX-evoked open channel block.

The ensemble-averaged currents obtained by averaging single channel responses (116 trials for 30 mM glycine, 202 trials for continuous 10 µM PTX) indicated a _decay similar to that observed for macroscopic glycine currents in the absence and presence of continuous PTX (Fig. 3). _decay was 131 and 31 ms, respectively, for the averaged currents in the absence and presence of continuous PTX (Fig. 4, A and B, bottom). Altogether these results indicate that the decrease in the deactivation time constant evoked by PTX could be mainly due to a decrease in the mean open time of the channel. If this hypothesis is true, increasing PTX concentration must decrease the mean open time in a concentration-dependent manner, as it does for the deactivation time constant of the glycine-evoked current (see Fig. 3B).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Acceleration of the decay phase of glycine-activated currents by the continuous presence of PTX. A1, current traces activated by 300 µM glycine (control), by co-application of 300 µM glycine and 10 µM PTX (plus 10 µM PTX), and by 300 µM glycine in the continuous presence of 10 µM PTX (plus continuous PTX) when PTX was maintained before, during, and after glycine application. Each trace represents the average of 12–15 responses. Note that the glycine-elicited currents in the control condition and with co-application of PTX returned slowly to the baseline after termination of glycine application, whereas the continuous presence of PTX eliminated the rebound current and significantly accelerated the decay phase. The thick line represents the application of 300 µM glycine. A2, normalized decay phase of glycine responses replotted on an expanded time scale to illustrate better the acceleration of the deactivation time course by continuous 10 µM PTX. The decay phase was best fitted with a single exponential function giving a decay time constant of 114 ms for control response, 116 ms for co-application of glycine and PTX, and 27 ms for continuous presence of PTX. Similar results were obtained from another 12 cells. B1, normalized responses evoked by short pulses (1 ms) of 30 mM glycine in the absence and presence of various concentrations of continuous PTX (shown beside each trace in micromolar). Note that the deactivation time constants decreased in a concentration-dependent manner. Each trace represents the average of 15–30 responses. B2, summary results for the deactivation time constants obtained from four different concentrations of PTX. ANOVA analysis indicated significant statistical differences (p < 0.01) among data groups. A and B were obtained from different patches.

To obtain an approximation of the binding rate constant for PTX, the reciprocals of the mean open times were plotted as a function of the PTX concentration as shown in Fig. 5D. Binding (k_on) and closing () rate constants were calculated from the relationship 1/_o = [PTX]k_on + (34), where _o is the mean open time and [PTX] is the PTX concentration. The linear fit to the data gave k_on = 11.6 µM^–1s^–1 and an value of 27.9 s^–1.

PTX Slows Down the Activation Kinetics of Glycine-evoked Current— The activation phase of current evoked by concentration steps of agonist gives important information on the kinetics of the receptor channels (23). The effects of PTX on the rising phase of macroscopic averaged currents evoked by glycine on ₂ homomeric GlyRs were therefore analyzed (Fig. 6). A series of 10–25 trials evoked with 10-s intervals was used to generate macroscopic currents as shown in Fig. 6A. The duration of the applications was adjusted to obtain a steady-state current. The rising phases of the outside-out currents evoked by the application of 300 µM glycine were best fitted with the sum of two exponential curves giving a fast rising time constant (_fast) and a slow rising time constant (_slow) as previously described (23). As shown in Fig. 6B, there was no significant difference in the _fast of currents activated by 300 µM glycine alone (_fast = 38 ± 5 ms, n = 14), during co-applications of glycine and PTX (_fast = 24 ± 5 ms, n = 13) or during the continuous application of PTX, before, during and after glycine application (_fast = 24 ± 4 ms, n = 12) (ANOVA, p > 0.05). _slow in the presence of co-application of 10 µM PTX and 300 µM glycine was significantly decreased, compared with responses evoked by glycine application alone (paired t test, p < 0.05). These results could suggest that PTX had little or no effect on glycine binding.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Decrease in mean open time in the continuous presence of PTX. A and B, representative, nonconsecutive, single-channel openings of a single 2 homomeric GlyR evoked by repetitive short pulses (1 ms) of 30 mM glycine in the absence (A) and presence (B) of continuous 10 µM PTX on the same patch (cut-off filter frequency, 2 kHz). The thick line represents the application of 10 µM PTX. Ensemble-averaged currents (lower traces; n = 116 for 30 mM glycine, n = 202 for +10 µM PTX) obtained from 7–9 different experiments were best fitted with a single exponential function (smooth lines). Decay time constants are indicated for both 30 mM glycine and +10 µM PTX. C and D, open time duration histograms obtained in the control (30 mM glycine, C), and in the continuous presence of 10 µM PTX (D) are shown as a function of log intervals with the ordinate on a square root scale. Histograms were better fitted with one exponential curve. Mean open time was decreased from 48.4 ms in the control condition to 6.1 ms in the continuous presence of PTX. E and F, closed time histograms in the control (30 mM glycine, E) and in the continuous presence of 10 µM PTX (F) were obtained. Histograms are shown as a function of log intervals, with the ordinate on a square root scale. The distributions were fitted with 1 and 2 exponential curves for the control and in the continuous presence of PTX, respectively. Note that c1 = 0.23 ms is very similar to the closed time constant c = 0.27 ms in the control conditions, whereas c2 = 5.76 ms is apparent in the continuous presence of PTX.

An Increase in the First Latency Accounts for the Increase in the Two Activation Time Constants—To determine if the increase in the two activation time constants of the rising phase of the currents that we observed in the presence of continuous application of PTX reflects changes in GlyR behavior occurring before channel conformational changes leading to the open state, we analyzed the distribution of initial closed times leading to the first opening (first latencies) in outside-out patches containing one active GlyR (see "Materials and Methods"). Fig. 7(A and B) shows the activation of a single receptor from the same patch in response to 200-ms step applications of 30 mM glycine in the absence (Fig. 7A) or in the continuous presence (Fig. 7B) of 10 µM PTX (application frequency, 0.1 Hz). The ensemble-averaged current obtained by averaging single channel responses (229–266 trials) had time courses similar to those observed for macroscopic currents in the absence or presence of continuous application of PTX, as previously described (see Fig. 6). As shown for macroscopic currents, the ensemble-averaged currents also exhibit a biphasic rising phase with fast and slow components, which were considerably slowed down in the continuous presence of PTX. The increase in the activation time constants observed in the presence of PTX appeared to be related to an increase in the first latency duration. The first latency cumulative distributions of the activation of a single GlyR evoked by 30 mM glycine application in the absence or presence of 10 µM PTX are shown in Fig. 7 (C and D). The first latency distributions were best fitted by the sum of two exponential functions with time constants _fast = 0.24 ms (85%), _slow = 2.2 ms (15%) in the absence of PTX and _fast = 4.7 ms (61%), _slow = 18.9 ms (39%) in the presence of continuous PTX. The corresponding ensemble-averaged current exhibited a rising phase with _fast = 0.33 ms (90%), _slow = 2.4 ms (10%) in the absence of PTX and _fast = 5.1 ms (48%), _slow = 20.7 ms (52%) in the presence of PTX, indicating that the slower activation phase of the ensemble current in the presence of continuous PTX was related to changes in GlyR conformational closed states distal from the channel open state.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. PTX decreased the mean open time of GlyR in a concentration-dependent manner. A–C, open time duration histograms obtained in the control (30 mM glycine; A) and in the continuous presence of 3 µM PTX (B) and 10 µM PTX (C) are shown as a function of log intervals with the ordinate on a square root scale. Histograms were better fitted with one exponential curve. Note that mean open time was decreased by PTX in a concentration-dependent manner. D, the reciprocals of the mean open times were plotted as a function of the PTX concentration, and the binding (kon) and closing () rate constants were calculated from the relationship 1/o = [PTX]kon + , where o is the mean open time, and [PTX] is the PTX concentration. The linear fit to the data gave a kon value of 11.6 µM–1s–1 and value of 27.9 s–1. Mean open times were obtained by pooling single-channel currents (22–66 trials) in one to three different experiments for each concentration of PTX.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Slower onset of macroscopic currents activated by saturating concentration of glycine in the continuous presence of PTX. A, averaged traces of currents (n = 10–25) obtained from the same patch showing the activation phase of the responses evoked by 300 µM glycine, co-application of 300 µM glycine, and 10 µM PTX (+10 µM PTX), and by glycine in the continuous presence of 10 µM PTX (plus continuous PTX). B, summary of data (n = 12–14) obtained from the experiments shown in A. NS, not significant; *, p < 0.05. C, averaged traces of currents (n = 12–15) obtained from the same patch showing the activation phase of the responses activated by saturating concentration of 30 mM glycine, co-application of 30 mM glycine and 10 µM PTX, and by glycine in the continuous presence of 10 µM PTX. Note that the activation phase of the glycine response was slowed down in the continuous presence of PTX. D, averaged traces of currents (n = 12–25) obtained from the same patch showing the activation phase of the responses evoked by saturating concentration of 30 mM glycine in the absence and presence of continuous 10µM PTX, and by oversaturating concentration of 100 mM glycine in the absence and presence of continuous 10 µM PTX. Note that increasing glycine concentration to 100 mM does not change the activation time course of current response activated by 30 mM glycine in the continuous presence of PTX. E and F, summary of data (n = 8–12) obtained from the experiments shown in C and D (NS, not significant).

View larger version (32K): [in this window] [in a new window]   FIGURE 7. Increase in the first latency accounts for the slower onset. A and B, representative, nonconsecutive, single-channel openings of a single 2 homomeric GlyR evoked by repetitive 200-ms step applications of 30 mM glycine in the absence (A) and presence (B) of continuous 10 µM PTX on the same patch (cut-off filter frequency, 2 kHz). Ensemble-averaged currents (lower traces; n = 266 for 30 mM glycine, n = 229 for +10µM PTX) were best fitted with a bi-exponential function (smooth lines). Fast and slow time constants and their relative areas are indicated for both 30 mM glycine and +10 µM PTX. C and D, first latency distributions in the absence (C) and presence (D) of continuous application of 10 µM PTX. The first latency distributions were best fitted by the sum of two exponential functions (smooth lines). Fast and slow time constants and their relative weights are indicated for both 30 mM glycine and +10 µM PTX. Note that the time constants and their relative areas of the first latency distributions are identical to those of the ensemble average currents, indicating that the slower onset of the ensemble average current in the continuous presence of PTX was due to increase in first latency duration.

When 10 µM PTX was applied immediately before (time interval of <0.1 ms) a concentration step of a saturating concentration of glycine (10 mM), it did not change the amplitude of the glycine-evoked current or its rising phase (Fig. 8B). The activation time constants were _fast = 0.4 ± 0.04 ms (81 ± 5%) and _slow = 2.6 ± 0.7 ms (19 ± 5%) ms in control conditions, and _fast = 0.4 ± 0.04 ms (82 ± 9%) and _slow = 2.4 ± 0.2 ms (18 ± 9%) ms with PTX pre-treatment (n = 5). This was not significantly different (paired t test, p > 0.5). These data indicate that it is unlikely that PTX can bind to unliganded GlyR.

According to the results described above, the lengthening of the rise time we observed in the continuous presence of PTX is unlikely to be due to PTX binding to unliganded GlyR. Moreover, simultaneous application of PTX and glycine had no significant effect on the rise time of the outside-out current. The only possibility of the lengthening of the rise time we observed in the continuous presence of PTX is that PTX, when applied during the deactivation of the glycine-evoked currents, modifies the activation kinetics of the next response. This hypothesis implies that PTX preferentially binds to GlyR in the open state and that it can remain bound after glycine washout. A similar PTX inhibitory effect was recently described for mutated R271C homomeric ₁ GlyRs (14). To test this hypothesis we analyzed the effect of PTX on the rise time of successive responses evoked by glycine when this alkaloid was applied during the relaxation phase of the first response (PTX post-treatment). PTX was applied for 500 ms, which corresponds to the full time-course of the deactivation phase of the glycine-evoked current without PTX. For these experiments we selected patches with a large number of GlyRs. This allowed us to compare the rise time between individual traces. The activation time constants were measured for glycine responses evoked 60 s after PTX post-treatment. They were compared with the values obtained in control conditions. To determine if this effect of PTX was reversible, we analyzed the rise time of responses evoked 3 s after the second application of glycine alone. As shown in Fig. 8C, post-treatment with 10 µM PTX was sufficient to speed up the deactivation phase of glycine-evoked current. The current evoked by the application of glycine alone up to 60 s after the end of the glycine current plus PTX post-treatment had an amplitude similar to that of the control response (<5% decrease, n = 7). Surprisingly, this current had a significantly slower rise time than that of control (n = 7, paired t test, p < 0.01). This was due to slower rising time constants _fast and _slow and to an increase in the proportion of the slow component of the activation phase. The activation time course of these responses was well fitted by the sum of two exponential curves as in the control, but with time constants _fast = 2.2 ± 0.2 ms (38 ± 1%) and _slow = 31 ± 3 ms (62 ± 1%). Applying glycine after (3 s) the response with a slower rising phase (Fig. 8C) evoked a current with activation time constants very similar to control values: _fast = 0.5 ± 0.04 ms (85 ± 5%) and _slow = 3.1 ± 0.3 ms (15 ± 5%). These results clearly indicate that the lengthening of the rise time evoked by PTX can persist up to 60 s after washout of glycine and PTX. They also indicate that GlyRs must be reactivated to allow recovery from the PTX effect. It is therefore likely that PTX can be trapped at its binding site when the channel closes and/or when glycine molecules dissociate from their binding sites.

View larger version (25K): [in this window] [in a new window]   FIGURE 8. Channel re-openings were required for recovery from PTX block. A1, average of 5 traces of current obtained in response to a 600-ms step application of 10 mM glycine and transiently inhibited by a 300-ms step application of 10 µM PTX with 10 mM glycine. The dashed boxes in A1 indicate parts of the trace enlarged in A2 (left box) and A3 (right box). A2, the onset of the picrotoxin inhibition was well fitted by a mono-exponential curve (gray dashed line) giving a time constant of 12 ms. A3, the recovery from the inhibition by PTX was best fitted by a bi-exponential curve (gray dashed line) giving time constants fast = 4 ms (28%) and slow = 37 ms (72%). B1, average of 5 traces showing currents evoked by a 300-ms step application of 10 mM glycine following a 300-ms step application of control solution (left black trace) or 10 µM PTX (right gray trace). Dashed boxes in B1 indicate the part of the traces enlarged in B2. B2, the onset of the both responses was best fitted by a bi-exponential function. The fast and slow time constant values and their relative area were respectively indicated in black (control preincubation) and in gray (PTX preincubation). Note the absence of effect of the PTX preincubation. C1, example of three consecutives responses to 200-ms step application of 10 mM glycine where the first application was directly followed by a 500-ms step application of 10µM PTX. The delay between each application is indicated between each trace. Note the quickening in the decay of the first glycine response during the PTX application and the slowing down in the onset of the second response. The third response exhibits an onset similar to the first response indicating a complete recovery from PTX effect. Dashed boxes indicate the part of the two first traces enlarged in C2. C2, the onset of the first and second responses was best fitted by a bi-exponential function. The fast and slow time constant values and their relative area were respectively indicated in black (first application, mean of 5 traces) and gray (second application, mean of 5 traces).

In the PTX block models we envisioned, PTX binds within the vestibule of the channel, which shortens channel opening (A₃OtoA₃PB; see Fig. 9B). It is then trapped at its binding site when the channel goes back to its closed-state conformation (A₃PB to A₃PC, Fig. 9B). In these models glycine can unbind while PTX remains trapped.

Two PTX block model subtypes were tested. In model 1 (Fig. 9B), the only way for PTX to bind and unbind is from the open state. The second type of model (models 2 and model 3, Fig. 9, C and D) supposes that the PTX binding site is not fully masked when the channel is in its bound closed conformation. This is also the case for GABA_C receptors (17). In model 2, one step was incorporated between the fully glycine-liganded closed states (A₃C) and the corresponding glycine-liganded closed states plus PTX (A₃PC). Accordingly, this model contains one cyclic scheme (Fig. 9C). This model supposes that PTX can escape from its binding site only when GlyR is fully liganded. In model 3, PTX is trapped when the receptor goes back to its unbound closed state. In this model, three steps were incorporated between the glycine-liganded closed states (A₂ plus AC, A plus A₂C, and A₃C) and the corresponding glycine-liganded closed states plus PTX (A₂ plus APC, A plus A₂PC, and A₃PC). Accordingly, this model contains three cyclic schemes (Fig. 9D). This model is somewhat similar to the kinetic model proposed for GABA_C receptors (17).

To compare the different models accounting for PTX inhibition, we fitted experimental traces obtained by long application of 0.3 mM or 30 mM glycine in the presence of 1, 3, and 10 µM PTX (n = 12 patches). All rate constants estimated with the control model for GlyR were set as fixed parameters. For simplicity, the glycine association rate constant (k_on) linking the different glycine-bound states plus PTX (A₂ plus APC, A plus A₂PC, and A₃PC), the desensitization rate constants and the corresponding recovery rate constants linking the liganded closed states plus PTX and the desensitization states (A₂ plus APD, A plus A₂PD, and A₃PD) were also set as fixed variables. All other parameters were set as free variables. We imposed constraints depending on the model tested. Model 1 had no constraint, but models 2 and 3 must have constrained reactions depending on the reaction cycles to satisfy the principle of microscopic reversibility (34). In model 3, the on reactions and the off reactions linking the liganded closed states with and without PTX were set as equivalent (A₂ plus AC to A₂ plus APC; A plus A₂C to A plus A₂PC; and A₃CtoA₃PC, respectively). Such a simplification postulating that PTX affinity is similar for the three bound closed states of the receptor was also proposed for GABA_C receptors (17). It is however important to note that when these reactions were set as independent, the fit of the experimental traces was not improved and the rate constants for these steps diverged considerably.

View larger version (24K): [in this window] [in a new window]   FIGURE 9. Kinetic schemes used for fitting glycine responses in the absence and presence of PTX. A, agonist; P, PTX; C, resting states of the receptor; D, desensitized state; and O, open state. A, this kinetic scheme was used for homomeric 2 GlyR in control conditions (without PTX). B, in model 1, PTX can bind and unbind from the GlyR open state only. PTX remains bound if continuously applied when glycine dissociates from its binding sites. C, in model 2, PTX can bind and unbind from the fully glycine-liganded closed state or the open state of GlyR. In this scheme, PTX is trapped when glycine dissociates from the fully liganded closed state. D, in model 3, PTX can bind and unbind from all glycine-bound states, but PTX is only trapped when the receptor returns to the glycine-unbound closed state.

Model 2 (Fig. 9C) provided a better prediction of our experimental results. This model gave 5 ± 0.8 times significant lower SSEs value than model 1 (ANOVA, p < 0.01). Incorporating steps for PTX binding to the other liganded bound states (model 3; Fig. 9D) did not significantly improve the fit when compared with model 2 (ANOVA, p > 0.1), suggesting that, although such transitions could exist, they were not necessary to describe our experimental data. The optimal averaged rate constant values obtained for models 2 and 3 are listed in Table 1. As shown in Table 1, fits of experimental data with models 2 and 3 gave very similar values for PTX association and dissociation rate constants. In models 2 and 3, the affinity of PTX for the channel open state (model 2: koff1p/Kon1p = 9.6 µM; model 3: koff1p/Kon1p = 10.7 µM) was found to be lower than that for the bound closed state (model 2: koff2p/Kon2p = 1.6 µM; model 3: koff2p/Kon2p = 1.2 µM). When we attempted to set the affinity of PTX for the channel open state equal to that for the liganded closed states, the SSEs of the fit was 3.1 ± 0.7 times significantly higher (ANOVA, p < 0.01), suggesting that the rate constant values for these PTX binding steps are unlikely to be equivalent. A similar conclusion was reported for PTX inhibition of GABA_C receptors (17). Because channel gating must involve large conformational changes, it is reasonable to suppose that the access of PTX to its binding site will be different when the receptor is in a bound closed conformation and in a bound open conformation (17).

Fig. 10 shows examples of fits of experimental traces using model 2 (thick dark lines) to responses evoked by the co-application of 30 mM glycine and 10 µM PTX (Fig. 10A) or 0.3 mM glycine and 1, 3, and 10 µM PTX (Fig. 10B). The model predicts a stable current amplitude in the presence of 30 mM glycine and 10 µM PTX and a small rebound current at the end of the co-application of PTX and glycine occurring for PTX concentration 3 µM (Fig. 10, A and B). The model also predicts an increase in PTX IC₅₀ when glycine concentration is increased (Fig. 10C). Parameters listed in Table 1 predict a PTX IC₅₀ of 3.4 µM and of 9.3 µM in the presence of 0.3 mM glycine and 30 mM glycine, respectively. This is in good agreement with our experimental data (2.7 µM and 6.4 µM, respectively; Fig. 1B). When the rate constant for PTX association from the open state was set as a free parameter, it was close to 5 µM^–1 s^–1 (Table 1), which is in reasonably good agreement with our experimental measurements (11.5 µM^–1 s^–1). This model also predicts the acceleration of the relaxation phase of the glycine-evoked current observed in the continuous presence of PTX (Fig. 10D), the lengthening of the rise time of the 30 mM glycine-evoked current (Fig. 10F) during continuous application of PTX and the lack of PTX effect on the rise time of currents evoked by 0.3 mM glycine (Fig. 10E). It also predicts the lengthening of the rise time of the glycine-evoked current when PTX was applied during the deactivation phase of the preceding response (Fig. 10G). Overall, these data indicate that model 2 characterized by the presence of two PTX binding steps, one from the fully liganded closed state and one from the open state, is the minimal stochastic scheme that best predicts PTX inhibitory effects on homomeric ₂ GlyRs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION