Two Novel Residues in M2 of the gamma -Aminobutyric Acid Type A Receptor Affecting Gating by GABA and Picrotoxin Affinity*

Andreas BuhrDagger , Clemens WagnerDagger , Karoline Fuchs§, Werner Sieghart§, and Erwin SigelDagger

From the Dagger  Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland, and the § Brain Research Institute, Section for Biochemistry and Molecular Biology, University of Vienna, A-1090 Vienna, Austria

Received for publication, September 29, 2000, and in revised form, December 11, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An amino acid residue was found in M2 of gamma -aminobutyric acid (GABA) type A receptors that has profound effects on the binding of picrotoxin to the receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha 1beta 3 receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha 1 subunit with the mutated beta 3 subunit beta 3L253F led to spontaneous picrotoxin-sensitive currents in the absence of GABA. Nanomolar concentrations of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the inward current was indicative of a chloride ion selectivity. The amplitude of the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold decrease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighboring residue beta 3A252V resulted in receptors that displayed an about 6-fold increased apparent affinity to GABA and an about 10-fold reduced sensitivity to picrotoxin.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The GABAA1 receptors are the major inhibitory neuronal ion channels in the mammalian central nervous system. Two subunits termed alpha  and beta  have initially been purified from bovine brain (1) and the corresponding DNAs have been cloned (2). Many mammalian subunits have been cloned since (3-8). These subunits show a high degree of homology to subunits of the nicotinic acetylcholine receptors, the glycine receptor and and the serotonin (type 3) receptor. The GABAA receptor is the site of action of many drugs, among them the benzodiazepines (for review see Ref. 9). The binding site for the channel agonist GABA and that for benzodiazepines are thought to be located at subunit interfaces in homologous positions (for reviews see Refs. 10 and 11).

The second transmembrane segment of these subunits lines the ion channel. In GABAA receptor alpha 1 subunits, residues Val256, Thr260, Thr261, Leu263, Thr264, Thr267, Ile270, Ser271, and Asn274 have been reported to be exposed to the channel lumen (12). Picrotoxin is likely to interact with residue Val256 (13, 14). Mutation in a homologous residue in Drosophila receptors confers cyclodiene resistance (15). A leucine residue is strictly conserved in the middle of the M2 region of all subunit isoforms and is located at position 263 of the alpha 1 subunit. Substitution to a serine in alpha 1, beta 2, or gamma 2 resulted in an abnormally high apparent GABA affinity for channel opening (16). Some point mutations of this leucine on alpha 1, beta 1, beta 2, or rho 1 subunits resulted in spontaneous open channels (17-21).

During work aimed at the understanding of the site in M2 involved in the recognition of tert-butylbicyclophosphorothionate (22), we investigated the properties of chimeric alpha 1beta 3 receptor subunits coexpressed with alpha 1 subunits. The results indicated an importance of Ala252 and Leu253 for the tert-butylbicyclophosphorothionate binding affinity. Here, we show that substitutions of beta 3A252 and beta 3L253 result in reduced picrotoxin affinity. For beta 3L253F affinity is more than 100-fold reduced, and a huge transient opening of the channel upon removal of picrotoxin was evident. Such a transient channel opening has not been observed for any other mutation before, and our mathematical model suggests that this is a consequence of the strongly decreased affinity for picrotoxin. Residue Val256, neighboring Phe257, the homologue on the alpha 1 subunit, has been shown to covalently interact with other noncompetitive blockers acting at the picrotoxin binding site (14). Leucine 253 of the beta 3 subunit (beta 3L253) may therefore be part of the contact site for picrotoxin together with alpha 1V256. In addition, we report that single point mutation beta 3L253F confers abnormal gating properties to alpha 1beta 3 receptors. These include spontaneous opening of the channels and a very high GABA sensitivity for channel gating, Thus, our work points to the involvement in GABAA receptor channel gating of more N-terminally located amino acid residues than previously suggested.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amino Acid Residue Numbering-- Residues are numbered according to the mature rat sequences.

Construction of Receptor Subunits-- The cDNAs coding for the alpha 1, beta 3, and chimeric subunits of the rat GABAA receptor channel have been described elsewhere (22, 23). Site-directed mutagenesis was done using the QuikChangeTM mutagenesis kit (Stratagene). In vitro synthesized sequences have been verified by DNA sequencing.

Functional Expression and Characterization-- Xenopus laevis oocytes were prepared, injected, and defolliculated, and currents were recorded as described (24, 25). Briefly, oocytes were injected with 50 nl of capped, polyadenylated cRNA dissolved in 5 mM K-HEPES, pH 6.8. This solution contained the transcripts coding for the different subunits at concentrations of 75 nM. RNA transcripts were synthesized from linearized plasmids encoding the desired protein using the mMessage mMachine kit (Ambion) according to the recommendations of the manufacturer. A poly(A) tail of ~300 residues was added to the transcripts by using yeast poly(A) polymerase (Amersham Pharmacia Biotech). The cRNA combinations were coprecipitated in ethanol and stored at -20 °C. Transcripts were quantified on agarose gels after staining with Radiant Red RNA Stain (Bio-Rad) by comparing staining intensities with various amounts of molecular weight markers (RNA Ladder; Life Technologies, Inc.). Electrophysiological experiments were performed by the two-electrode voltage clamp method at a holding potential of -80 mV. GABA and picrotoxin (Fluka) were applied for 20 s, and a washout period of 3-15 min was allowed to ensure full recovery from desensitization. The perfusion solution (6 ml/min) was applied through a glass capillary with an inner diameter of 1.35 mm, the mouth of which was placed about 0.4 mm from the surface of the oocyte. The rate of solution change under our conditions has been estimated 70% within less than 0.5 s (25). Current responses have been fitted to the Hill equation: I = Imax/(1+(EC50/[A])n) where I is the peak current at a given concentration of GABA (A), Imax is the maximum current, EC50 is the concentration of agonist eliciting half-maximal current, and n is the Hill coefficient. Currents were measured using a modified OC-725 amplifier (Warner Instruments Corp.) in combination with a xy recorder or digitized using a MacLab/200 (AD Instruments).

Kinetic Modeling-- We analyzed the gating behavior of the mutated channel using a kinetic model that omits all states that only become marginally populated (26). For arguments described later, the model consists of four states: two different states for closed channels (C, R), one state for open channels (O) and one state for channels blocked by picrotoxin (O.PTX). The triangular three state model in the absence of picrotoxin (O, C, R) requires six microscopic rate constants. They are calculated using the experimentally determined current decay constants from the open to the closed states (tau 1 and tau 2), the equilibrium constant for the spontaneously open state, and the law of detailed balancing. The remaining rate constants were used to adjust the simulation to the data. The binding of picrotoxin to the receptors adds four new rate constants to the system. They have to provide the reopening constant measured in the presence of picrotoxin (tau ), and they have to satisfy the law of detailed balancing. The additional two rates were used to fit the data. Therefore, from the 10 rate constants in total there are only four that can be used to reconcile the simulations with the data. The kinetics of application and washout of picrotoxin is not taken into account. They are assumed to happen instantaneously. This assumption is justified because the processes studied here develop at a much slower time scale. The integration of the differential equation system was performed using the subroutines of the Matlab 5.2.0 library (MathWorks Inc.).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Receptors Containing Chimeric Subunits-- Fig. 1 shows the structure of wild type and chimeric subunits. The chimera consist of N-terminal sequences of the beta 3 subunit fused to C-terminal sequences of the alpha 1 subunit. They differ from each other in the residues forming the ion channel pore region. Each of the chimeras was coexpressed together with the alpha 1 subunit in Xenopus oocytes. Mature receptors incorporated in the surface membrane were analyzed by using the two-electrode voltage clamp technique. Heteromeric alpha 1CH7 receptors failed to respond to GABA (<10 nA), and an apparent outward current could be measured during application of 1 mM picrotoxin alone (Fig. 2A). Interestingly, a strong transient inward current was detected during washout of picrotoxin in the absence of GABA (Fig. 2A). Wild type alpha 1beta 3 receptors were activated by GABA and showed no response to the application of picrotoxin alone (Fig. 2B). Injection of cRNA coding for CH7 alone did not result in ion currents induced by picrotoxin or GABA (data not shown), indicating that the channel with these unusual properties was formed from alpha 1CH7. Three additional chimeras were coexpressed together with the alpha 1 subunit to localize residues important for the unusual gating behavior. Only alpha 1CH74 also displayed the picrotoxin washout current found in alpha 1CH7 receptors (Table I). An amino acid comparison between the chimera revealed that a threonine-valine-phenylalanine motif was common to CH7 and CH74 and absent in the other two constructs. Three mutant subunits were constructed by individually introducing these three residues of alpha 1 into the homologous positions of beta 3, with the aim to identify the amino acid residue responsible for the abnormal properties.



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Fig. 1.   Schematic drawing of the alpha 1, the beta 3, and chimeric subunits. N, N terminus; C, C terminus; boxes represent transmembrane regions M1-M4. alpha 1 sequences are represented by thin line and open boxes. beta 3 sequences are represented by a thick line and gray boxes. CH7 is composed of the N-terminal amino acids 1-250 of the beta 3 subunit and the amino acids 255-428 of the alpha 1 subunit. The amino acid sequences of the M2 regions of alpha 1 and beta 3 are compared. The position of leucine 253 in the beta 3 subunit is marked by an asterisk. A schematic representation of the M2 regions of wild type and chimeric subunits is drawn in the lower part of this figure. The chimera consist of the N terminus of the beta 3 subunit fused to the C terminus of the alpha 1 subunit and differ from each other by the sequences present in the M2 regions. The upper part of the figure is taken from Ref. 22.



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Fig. 2.   Electrophysiological properties of alpha 1CH7 and of alpha 1beta 3 receptors. Currents were recorded under voltage clamp conditions at -80 mV from oocytes injected with alpha 1 and CH7 (A), with alpha 1 and beta 3 (B), or with alpha 1 and beta 3L253F (C) subunits. 1 mM GABA and 1 mM picrotoxin (Ptx) were applied for 20 s each (A and B, bars) or 1 min (C). Two additional identical experiments in each case gave similar results. The same behavior of the channels was observed by using lower concentrations of picrotoxin in three additional experiments and shorter application times in 17 additional experiments.


                              
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Table I
Properties of wild type and chimeric receptors
Responses to 1 mM GABA or 1 mM picrotoxin are shown.

Expression of Point Mutated Receptors-- Wild type and mutant beta 3 subunits were coexpressed together with the alpha 1 subunit in Xenopus oocytes. All three mutant receptors expressed GABA-activated chloride currents. Receptors with a leucine to phenylalanine substitution in the channel pore-forming region at position 253 (alpha 1beta 3L253F) could be activated by very low concentrations of GABA (30 nM). Repeated applications elicited increasingly smaller current amplitudes even when a washout period of up to 15 min was used (data not shown). For this reason it was technically not possible to measure the apparent affinity of GABA for channel opening in alpha 1beta 3L253F. Dose response curves for wild type alpha 1beta 3, alpha 1beta 3V251T, and alpha 1beta 3A252V revealed 3.5- and 6.0-fold increases in the apparent affinities to GABA for these mutated receptors (Table II), respectively.


                              
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Table II
Properties of wild type and mutant GABAA receptor channels

Response to Picrotoxin-- Application of 1 mM picrotoxin in the absence of GABA did not induce any apparent outward or inward currents in alpha 1beta 3V251T and alpha 1beta 3A252V receptors. In contrast, in oocytes expressing alpha 1beta 3L253F receptors, picrotoxin application resulted in apparent outward currents (Fig. 2C) similarly to oocytes expressing alpha 1CH7. The mutated channel alpha 1beta 3L253F also showed a huge transient inward current upon washout of picrotoxin (Fig. 2C). At 1 mM picrotoxin, the amplitude of the transient inward current was 11 times (average of three determinations) the amplitude of the apparent outward current.

To exclude that the current properties upon injection of cRNAs coding for alpha 1 and beta 3L253F resulted from homomeric beta 3L253F, we also expressed this mutated subunit alone. No detectable signal could be obtained with 300 µM picrotoxin (two independent batches of oocytes).

A voltage ramp protocol was used to measure ion currents in mutant alpha 1beta 3L253F channels before and during and the application of picrotoxin and about 5 s after its removal (Fig. 3A). The voltage ramp had a duration of 0.13 s, such that the amplitude of the inward current showed little change during the application of the ramp. Picrotoxin application resulted in a reduction of the membrane conductance, indicating that part of the receptors are spontaneously in an open conformation. All three curves obtained had the same intersection at -38 ± 1 mV (three experiments). Therefore, it can be concluded that the spontaneous current and the transient inward current have the same ion permeability. Replacing the 95 mM Cl- with 9.5 mM Cl- and 84.5 mM acetate- in the outside medium resulted in an about 60 mV shift to the right in the reversal potential of the transient inward current (not shown), in line with a chloride selective conductance. From the reversal potential of -38 mV determined at an extracellular chloride concentration of 95 mM, an intracellular chloride concentration of ~23 mM may be estimated. The same voltage ramp protocol was used before and during the application of a subsaturating concentration of GABA to oocytes expressing the wild type alpha 1beta 3 receptor (Fig. 3B). The intersection of the two curves was found at -25 ± 2 mV (three experiments), indicating an intracellular chloride ion concentration of about 37 mM. The difference in the intracellular chloride concentration in oocytes expressing wild type and the point mutated receptor can be explained by a more negative membrane potential of the oocyte than the chloride reversal potential. Under conditions where the permeability for this ion is increased, the membrane potential drives chloride ions out of the cell.



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Fig. 3.   Voltage dependence of ion currents of alpha 1beta 3L253F (A) and alpha 1beta 3 (B) receptors. Continuous voltage ramps of 130 ms duration were generated from a holding potential of -80 to +50 mV, and ion currents were recorded. The experiment was first performed in the absence of picrotoxin (medium), then in the presence of 0.3 mM picrotoxin, and finally during the washout of the drug in A and first during perfusion with medium and then during perfusion with medium containing 0.3 µM GABA in B. Two additional experiments in each case gave similar results.

As mentioned above, the channel opens to a certain degree spontaneously producing an inward current. The initial response to the application of picrotoxin is an apparent outward current reflecting channel closure. The picrotoxin concentration dependence of the peak current amplitude is illustrated in Fig. 4A. It increases between 10 and 1000 µM picrotoxin and shows no saturation up to 1 mM picrotoxin (Fig. 4C). The maximum apparent outward current may, however, be estimated, assuming an infinite membrane resistance for the case where all channels are in the closed state. Assuming that most channels are in the open state upon removal of picrotoxin, it can be estimated that less than 9% of the channels are spontaneously open prior to the application of picrotoxin. The shape of the late apparent outward current response during perfusion with picrotoxin depends on the concentration of picrotoxin used (Fig. 4A). In the presence of low concentrations, only a transient inhibition of the current could be detected, followed by reopening of the channels. The time course of this reopening seemed picrotoxin concentration independent in the range of 10-300 µM. Because of the small amplitudes of this component, it could only be estimated. Assuming a mono-exponential time course reopening was characterized by a tau  in the range of ~6-13 s (not shown).



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Fig. 4.   The size of the apparent outward current and the transient inward current in alpha 1beta 3L253F receptors both depend on the concentration of picrotoxin. A, current traces obtained by applying increasing concentrations of picrotoxin (bars) for 1 min. B, computer simulations describing the concentration dependence of the transient inward current evoked by picrotoxin withdrawal in the mutated alpha 1beta 3L253F GABAA receptor. The assumed concentrations of picrotoxin were 10, 30, 100, 300, and 1000 µM. The application time was 60 s. The rate constants are described in the legend to Fig. 7 and assume the following values: k1 = 5.0 × 10-2 µM-1 s-1, k2 = 1.5 s-1, k3 = 8.1 × 10-2 s-1, k4 = 8.1 × 10-3 s-1, k5 = 3.0 × 10-4 µM-1 s-1, k6 = 1.0 × 10-1 s-1, k7 = 5.0 × 10-2 s-1, k8 = 5.5 × 10-2 s-1, k9 = 1.0 × 10-3 s-1, and k10 = 9.0 × 10-3 s-1. C, size of the apparent outward current amplitudes and the inward current amplitudes from three different oocytes. Data are given as the means ± S.D.

A large transient inward current was observed during washout of picrotoxin. The concentration dependence on picrotoxin of this current is illustrated in Fig. 4C. Its amplitude increases using increasing concentrations of picrotoxin and does not saturate up to 1 mM picrotoxin. Half of the amplitude observed at 1 mM picrotoxin was observed at about 300 µM (Fig. 4C). Fig. 5A shows that the size of this current increases with the duration of picrotoxin application. For this experiment 300 µM picrotoxin was applied during different time intervals between ~1 and 60 s. The time dependence of the increase in inward current amplitude is well fitted with a mono-exponential function with tau  = 9.1 ± 2.2 s (n = 3). Reclosure of the channel after transient opening was independent of the picrotoxin concentration and followed a bi-exponential time course with tau f = 5.5 ± 2.8 s and tau s = 23.3 ± 12.0 s (means ± S.D., three experiments at five different concentrations each).



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Fig. 5.   The size of the washout current depends on the duration of picrotoxin application in alpha 1beta 3L253F receptors. A, 300 µM picrotoxin was applied for ~1, ~2, 4, 8, 16, 30, and 60 s (bars). Two additional experiments gave similar results. B, computer simulation of the dependence of the inward current on the time span of picrotoxin application. Duration of 300 µM picrotoxin perfusion increases from 1 s to 2, 4, 8, 16, 30, and 60 s. The rate constants were as described in the legend to Fig. 4B.

The picrotoxin sensitivity of GABA-activated currents was also measured for wild type alpha 1beta 3 receptors and mutant alpha 1beta 3V251T and alpha 1beta 3A252V receptors. A GABA concentration eliciting 10-15% of the maximum current was used in these experiments. Because alpha 1beta 3V251T and alpha 1beta 3A252V receptors displayed an increased apparent affinity to GABA (Table II), a lower concentration of agonist had to be used for these two mutant receptors. alpha 1beta 3A252V receptors displayed an about 10-fold reduced sensitivity to picrotoxin compared with wild type and alpha 1beta 3V251T receptors (Fig. 6).



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Fig. 6.   Decreased picrotoxin sensitivity of GABA-activated currents in alpha 1beta 3A252T receptors. A, current traces in the absence and in the presence of increasing concentrations of picrotoxin (Ptx, upper bars). 1 µM GABA was used for oocytes expressing wild type receptors, and 0.3 µM GABA was used for oocytes expressing alpha 1beta 3V251T or alpha 1beta 3A252V (lower bars). B, relative current amplitudes at the end of 20 s of picrotoxin application. Data are given as the means ± S.D. of three experiments.

Kinetic Modeling-- To explain the following two phenomena, namely the reopening of the channels during continuous application of picrotoxin and the transient inward current after its removal, we performed computer simulations based on the model shown in Fig. 7. Rate constants used are given in the figure legends of Figs. 4B. It is assumed that about 5% of the channels are spontaneously open, which determines the equilibrium state of the system and the initial conditions for the simulations. The channels switch between this open state and the closed states giving rise to the observed inward resting current. Washout of picrotoxin is assumed to lead to fast dissociation from the receptor without rebinding. This results in the immediate emptying of all bound states. The number of picrotoxin less closed states is then given by the relaxation process upon picrotoxin removal. The double exponential decay determined in the experiments requires a minimal model which consists of three states: an open state (O) and two closed states (C and R). Applying picrotoxin to the system leads to a pronounced reduction in the number of open channels. The resulting initial decrease of the inward current (apparent outward current) is followed by the relaxation into a new equilibrium state. We propose that only the state (O.PTX) (binding of picrotoxin to the open channels) is kinetically relevant because after withdrawal of picrotoxin these channels are able to produce a transient inward current. If in contrast picrotoxin binding to closed channels would be dominant, no transient inward current could be observed, because the channels would already be closed. Fig. 5B presents simulations of experiments where the duration of 300 µM picrotoxin application is varied. Because the equilibrium is on the side of the state (O.PTX) because of the excess of picrotoxin, the longer the transient filling of the state (O.PTX), the larger the transient inward current amplitude after removal of picrotoxin. Fig. 4B shows simulations of the current during perfusion with different concentrations of picrotoxin. The amplitude of the initial apparent outward current is limited at high picrotoxin concentration because of saturation of channel closure. The following relaxation process into the new equilibrium state depends only weakly on the concentration of picrotoxin (largest relaxation constant tau  = 13-16 s), which is in agreement with the experiments. The simulations revealed that the closed state (R) serves as a reservoir for states C and O so that after binding of the channels to picrotoxin the two latter states become partly refilled by channels from state R. Obviously the higher the concentration of picrotoxin, the larger the population of the state (O.PTX), and as a consequence the amplitude of the rebound current augments with increasing picrotoxin concentration. The true k2 that describes the transition rate from O.PTX to O is obscured by the rate of solution change (see "Experimental Procedures"). The model is constructed in such a way that the decay of the inward current after removal of picrotoxin is double exponential with time constants tau 1 = 8 s and tau 2 = 12 s. Both, the picrotoxin concentration dependence (Fig. 4B) and the time dependence (Fig. 5B) of the responses of the mutated channel to picrotoxin exposure and removal predicted by the model should be compared with the experimentally observed behavior (Figs. 4A and 5A). Except for the initial peak of the apparent outward current predicted from the model and almost absent in the experimental traces, the agreement between model and experiment is remarkable. The difference is probably mostly due to the limited rate of solution change (see "Experimental Procedures"), which has not been taken into account in the simulations.



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Fig. 7.   Model used in the computer simulations. The states O, C, R, and O.PTX denote the open state, the closed state, the resting state, and the state where picrotoxin is bound to the open conformation, respectively. Note that the state O.PTX is nonconducting because of the channel blocker picrotoxin. The value of the rate constants are given in the legend to Fig. 4B.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We studied alterations in the channel lining part of the recombinant alpha 1beta 3 GABAA receptor. A mutant ion channel was found that displayed a transient chloride current upon removal of the channel blocker picrotoxin. A single point mutation is able to confer this unusual property to the receptor, namely, of leucine 253 in the M2 region of the beta 3 subunit to a phenylalanine, which is present in the homologous position of all alpha  subunits. The point mutated beta 3 was coexpressed together with the alpha 1 subunit. These receptors can open spontaneously in the absence of GABA and are blocked by picrotoxin in a dose-dependent fashion. Washout of picrotoxin results in a strong transient inward current. The amplitude of this current is dependent on the concentration of picrotoxin and the time of its application. The point mutated receptors could be activated by very low concentrations of GABA. Obviously, this mutation strongly affects gating of the channel and its interaction with picrotoxin.

It is interesting to compare the newly identified position with other positions within M2 that are important for receptor function and modulation. Based on radioligand binding experiments to chimeric receptors, it has been suggested that beta 3L253 is important for the binding of another channel blocker tert-butylbicyclophosphorothionate (22). This position is 12 amino acid residues N-terminal to the one on the beta 3 subunit implicated in the action of loreclezole (27). It is located 6 residues C-terminal to the predicted cytoplasmic entry point into the membrane and is 6 residues N-terminal to the conserved leucine in the center of the M2 region. Point mutations of this leucine also resulted in spontaneous currents (17-20). The spontaneous currents were in some cases antagonized by GABA (18, 19) and were sensitive to picrotoxin (18-21). Spontaneous channel activity has also been reported with expression of the rat or mouse beta 1 subunit alone (28, 29), the mouse beta 3 subunit alone (30), and a combination of rat alpha 5 and beta  subunits (31). Using the mentioned amounts of cRNA, no spontaneous currents were observed for homomeric wild type beta 3 and mutant beta 3L253F receptors. In all the mentioned cases an off-current upon picrotoxin washout was never reported. Our model presented below will predict why this is not the case.

The structure of the GABAA receptor in M2 is presumably close to that of other members of the ligand-gated ion channel family. The above cited studies and studies of the homologous nicotinic acetylcholine receptor suggested that the centrally conserved leucine is structurally critical for channel gating and might even be a part of the channel gate (32). Other studies place the gate more N-terminal to the cytoplasmic end of the channel pore (13). Because alpha 1beta 3L253F receptors display an altered channel gating our results support this proposal.

Valine 256 on the alpha 1 subunit has been proposed to be exposed to the channel lumen (13) and to be in direct contact with picrotoxin (13, 14). A homologous residue has also been implicated in the action of the cyclodiene insecticide resistance in invertebrate GABA receptor subunit Rdl (15). Because of the 5-fold symmetry of the channel pore, it is likely that the homologous residue on the beta 3 subunit, alanine 252, is also facing the channel pore and might be close to the bound picrotoxin entity. Our results indeed show that replacement of alanine by valine in this position (beta 3A252V) results in an about 10-fold reduced affinity for picrotoxin. Mutation of the adjacent leucine 253 to phenylalanine even more drastically reduces the picrotoxin affinity. It cannot completely be ruled out that the substitution might have some indirect effects on the picrotoxin binding pocket. However, it is tempting to speculate that picrotoxin is in direct contact with beta 3L253. In apparent contrast to this speculation, Xu and Akabas (12) found that the homologous phenylalanine 257 on the alpha 1 subunit after cysteine substitution is not accessible to sulfhydryl reagents. However, this residue is on the same side of the alpha -helix as reactive positions and is also adjacent to them. Side chains other than cysteine might be at least partially accessible to the channel lumen and could be in contact with the picrotoxin molecule.

We propose a kinetic model for alpha 1beta 3L253F describing its interaction with picrotoxin. It makes a number of predictions that are discussed in the following. Mutant alpha 1beta 3L253F receptors assume an open and two closed states in the absence of GABA. The open state is inferred from the presence of spontaneously open channels, and the closed states are required because of the double exponential decay of the transient inward current upon picrotoxin removal. Picrotoxin is mostly bound to open channels that become nonconducting because of the bound channel blocker resulting in an apparent outward current. Reopening of channels in the presence of picrotoxin finds a simple explanation with the help of our model. The binding of picrotoxin to the open (O) and closed channel (C) is faster than the transition between the two closed states (R and C). After emptying states O and C by adding picrotoxin, the channels in the desensitized state R serve as a reservoir that slowly releases channels to the open state via state C. The equilibrium of this process is reached in about 1 min (Figs. 4 and 5). The regain of open channels is responsible for the increase of the inward current in the presence of picrotoxin.

The huge transient inward current after picrotoxin washout is the result of the decreased affinity (fast transition of O.PTX to O), and it depends on both the duration of picrotoxin exposure and on its concentration (Figs. 4 and 5). Because of the almost instantaneous transition of O.PTX to O upon picrotoxin removal, the amplitude of the initial washout current is approximately proportional to the population of state O.PTX. Therefore, we can use this amplitude to estimate the overall equilibrium affinity constant to picrotoxin. It is higher than 300 µM in the present case for alpha 1beta 3L253F receptors, although the local affinity constant of the reaction between O and O.PTX is 20 µM in our model. The former value should be compared with 3 µM picrotoxin that reduces GABA induced currents in alpha 1beta 3 to about 50% (Fig. 4C).

As mentioned above, substitution of the centrally conserved leucine in GABA receptors in some cases also resulted in the formation of spontaneously open channels. Our model shows that the large amplitude of the fast transient inward current requires a faster transition from O.PTX to O as compared with all other transition rates of the model. Increasing the time constant for this process reduces the amplitude and slows down the time course for the transient population of the open state. Both effects are observed for other GABAA receptors with substitution of the centrally conserved leucine (18-21). Therefore, the model suggests that for these mutated GABAA receptors the dissociation of picrotoxin from the blocked channel (k2) is slowed down. The relative amplitude of this fast transient inward current is also limited by the fraction of channels that are in the open state in the absence of picrotoxin. The larger this fraction of spontaneously open channels is, the smaller the predicted inward current. Both factors may contribute to a different degree to the lack of an transient inward current in the initially cited cases of spontaneous currents.

In summary, we report two important observations. The first is that we describe two point mutations in the M2 region of the beta 3 subunit close to the putative cytoplasmic end that both result in an increase of apparent GABA affinity. One of these, beta 3L253F results additionally in spontaneous channel opening and is thus structurally critical for channel gating. Our results therefore strongly suggest that the region important for channel gating has to be extended at least four amino acid positions toward the N terminus as compared with previous conclusions (18). The second observation is that these point mutations result in a reduced affinity for picrotoxin. The residue alpha 1V256 has been shown to covalently interact with other noncompetitive blockers acting at the picrotoxin binding site (14). Mutation of the homologous residue beta 2A252 shows a 10-fold effect. However, mutation of the neighboring residue beta 3L253 has drastic effects on picrotoxin affinity and may therefore together with alpha 1V256 form part of the contact site for picrotoxin.


    ACKNOWLEDGEMENTS

We thank Dr. F. Jursky (Bratislava) for contributions to the construction of the chimera and R. Baur (Bern) for help especially in microinjection of Xenopus oocytes.


    FOOTNOTES

* This work was supported by Grants 3100-05359998/1 from the Swiss National Science Foundation and European Union Grant BIO4-CT96-0585 (BBW 960010).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Bern, Friedbühlstr. 49, CH-3010 Bern, Switzerland. E-mail: Erwin.Sigel@pki.unibe.ch.

Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M008907200


    ABBREVIATIONS

The abbreviations used are: GABA, gamma -aminobutyric acid; GABAA, GABA type A.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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