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0026-895X/97/040676-07$3.00/0
Copyright © by The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY 52:676-682 (1997).

Residues at Positions 206 and 209 of the alpha 1 Subunit of gamma -Aminobutyric AcidA Receptors Influence Affinities for Benzodiazepine Binding Site Ligands

Andreas Buhr, Martin T. Schaerer, Roland Baur, and Erwin Sigel

Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland

    Summary
Summary
Introduction
Materials & Methods
Results
Discussion
References

Ligands of the benzodiazepine binding site allosterically modulate gamma -aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha  and gamma  subunits. We transiently expressed wild-type alpha 1beta 2gamma 2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alpha Y209A showed ~40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alpha Y209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta -carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alpha Y209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alpha T206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta -carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha 206 and alpha 209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha  helix.

    Introduction
Summary
Introduction
Materials & Methods
Results
Discussion
References

The GABAA receptor is one of the major inhibitory neuronal ion channels in mammalian brain. Two subunits were initially purified (1), and their coding DNA was cloned (2). A total of 14 mammalian subunits were subsequently cloned (3) (see Refs. 4-8 for reviews). The subunits show homology to subunits of the nicotinic acetylcholine receptor, glycine receptor and serotonin3 receptor.

The GABAA receptor is also the site of binding of benzodiazepines and related compounds (see Ref. 7 for review). Both the beta  and alpha  subunits are important for interaction with the channel agonist GABA (9-11). In addition to the well established importance of an alpha  subunit, the presence of a gamma  subunit is indispensable for benzodiazepine stimulation of GABA-induced currents (12, 13). Three amino acid residues in the gamma 2 subunit affect benzodiazepine pharmacology (14-17). Both alpha  and gamma  subunits are thought to contribute to the benzodiazepine binding site, but its localization remains unknown.

We recently described three point mutations in the alpha  and gamma  subunits that individually affect benzodiazepine effects in GABAA receptors on expression in Xenopus laevis oocytes (15). All of the mutated channels respond ~3-fold to diazepam compared with the wild-type. Two point mutations also result in channels with an enhanced response to zolpidem, whereas the mutation in the gamma  subunit results in a loss of the zolpidem effect (15). The loss in sensitivity toward zolpidem is accompanied by a loss in sensitivity toward alpidem, Cl 218872, and zopiclone (16). Although the benzodiazepine antagonist Ro 15-1788 can counteract diazepam effects, zolpidem cannot, indicating that zolpidem has lost its ability to interact with the receptor; direct binding studies on GABAA receptors transfected in HEK 293 cells confirmed this (16). Interestingly, some of the amino acid residues important for allosteric modulation by benzodiazepines are directly homologous to amino acid residues on other subunits implicated in the formation of the receptor agonist binding site.

In a previous functional study, the mutated amino acid Y209A of the alpha  subunit gave varying results with different ligands of the benzodiazepine binding site. Although there was a significant functional difference between wild-type channels and channels containing this mutated subunit toward 1 µM zolpidem, this divergent behavior was not significant for 1 µM diazepam. To decide whether this amino acid residue is important for the binding of ligands of the benzodiazepine binding site, we investigated the binding properties of the respective receptors after transient expression in HEK 293 cells. We analyzed two additional point mutants of this amino acid residue and the T206A mutant of the alpha  subunit. Results obtained suggest involvement of both amino acid residues alpha 206 and alpha 209 in the formation of the binding pocket for ligands of the benzodiazepine binding site. The positional difference of the two residues is suggestive of an alpha  helix.

    Materials and Methods
Summary
Introduction
Materials & Methods
Results
Discussion
References

Transfection of recombinant GABAA receptors in cultured cells. The cDNAs coding for the alpha 1, beta 2, and gamma 2 subunits of the rat GABAA receptor channel and the construction of point mutants have been previously described (18-20). HEK 293 cells were maintained in minimum essential medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin through standard cell culture techniques. Equal amounts (total of 20 µg of DNA/90-mm dish) of GABA receptor subunits were transfected into HEK 293 cells (American Type Culture Collection, Rockville, MD) through the calcium phosphate precipitation method (21). After overnight incubation, the cells were washed twice with serum-free medium and fed again with medium.

Membrane preparation. At ~60 hr after transfection, the cells were harvested by washing with ice-cold phosphate-buffered saline, pH 7.0, and centrifuged at 150 × g. Cells were washed with buffer containing 10 mM K phosphate, 100 mM KCl, and 0.1 mM K-EDTA, pH 7.4. Cells were homogenized through sonication in the presence of 10 µM phenylmethylsulfonyl fluoride and 1 mM EDTA. Membranes were collected in three centrifugation-resuspension cycles (100,000 × g for 20 min), and then used for ligand binding or stored at -20°.

Binding assays. Resuspended cell membranes (0.5 ml) were incubated for 90 min on ice in the presence of [3H]muscimol (26 Ci/mmol), [3H]Ro 15-1788 (87 Ci/mmol), or [3H]flunitrazepam (86 Ci/mmol) (Dupont-New England Nuclear, Boston, MA) and various concentrations of competing ligands. Nonspecific binding was determined in the presence of 25 µM unlabeled muscimol, Ro 15-1788, or flunitrazepam, respectively. Membranes (20-50 µg of protein/filter) were collected through rapid filtration on GF/C filters presoaked in 0.3% polyethyleneimine. After three washing steps with 4 ml of buffer, the filter-retained radioactivity was determined by liquid scintillation counting. On the basis of IC50 determinations, the Ki value was estimated according to the Cheng-Prusoff equation (22).

Detection of GABAA receptors on the surface of living, transfected HEK 293 cells. Culture medium was removed from transfected cells. Rabbit polyclonal antibodies raised against a polypeptide representing amino acid residues 1-9 of the rat GABAA receptor alpha 1 subunit (5 µg/ml) (23, 24) and dialyzed, tetramethylrhodamine isothiocyanate-coupled swine anti-rabbit IgG antibody (R 0156, 1:20; DAKO, Carpinteria, CA) were added in succession, each diluted in buffered saline and incubated for 45 min at room temperature. The cells were visualized using a Zeiss Axiovert 35 fluorescence microscope equipped with a 63× objective or on a confocal microscope (Zeiss LSM 100). Where indicated, cells were fixed using 4% paraformaldehyde before immunostaining.

Detection of alpha 1 GABAA receptor subunits on Western blots. Protein (15 µg) from transfected cells was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane. After blocking for 1 hr with 5% nonfat dry milk/0.05% Tween-80, lanes were successively subjected for 3 hr to 5 µg/ml concentrations of the above-mentioned rabbit primary antibody (25) and peroxidase-coupled anti-rabbit IgG antibody (R-14745, 1:200, 1 hr; Transduction Laboratories, Lexington, KY). Peroxidase activity was visualized by the ECL reaction (Amersham, Arlington Heights, IL).

Expression and functional characterization. X. laevis oocytes were prepared, injected, and defolliculated, and currents were recorded as previously described (13, 26). Briefly, oocytes were injected with 50 nl of cRNA dissolved in 5 mM K-HEPES, pH 6.8. This solution contained the transcripts coding for the different subunits at a concentration of 10 nM for alpha 1, 10 nM for beta 2, and 100 nM for gamma 2 (calculated from the UV absorption). Electrophysiological experiments were performed by the two-electrode voltage-clamp method at a holding potential of -80 mV. GABA dose-response curves were fitted using a least-squares method (Gauss-Newton-Marquardt). The equation used was I(c) = Imax cn/(cn + Kan), where c is the GABA concentration, I is the current elicited, Ka is the GABA concentration eliciting half-maximal current amplitudes (50% Imax), and n is the Hill coefficient. Each of the curves was then standardized to Imax = 100%, and the averaged data were fitted again. Allosteric potentiation via the benzodiazepine site was measured at a GABA concentration eliciting 5-15% of the maximal GABA current amplitude by application of GABA alone and coapplication of GABA with increasing concentrations of flunitrazepam. GABA was applied for 20 sec, and a washout period of 4 min was allowed to ensure full recovery from desensitization. Stimulation of GABA currents was expressed in percentage of the respective control current amplitudes and then standardized, taking the stimulation by 1 µM flunitrazepam of wild-type receptors (328%) as 100%. The perfusion system was cleaned by washing with dimethylsulfoxide to avoid contamination.

    Results
Summary
Introduction
Materials & Methods
Results
Discussion
References

Expression of wild-type and mutant receptors in HEK 293 cells. Wild-type alpha 1beta 2gamma 2 (hereafter referred to as alpha beta gamma ) were mutated in the tyrosine residue alpha 209 to alanine (alpha Y209Abeta gamma ), phenylalanine (alpha Y209Fbeta gamma ), or glutamine (alpha Y209Qbeta gamma ) or the threonine residue alpha 206 to alanine (alpha T206Abeta gamma ). Wild-type and point mutant alpha Y209Abeta gamma , alpha Y209Fbeta gamma , alpha Y209Qbeta gamma , and alpha T206Abeta gamma receptors were expressed by transient transfection in HEK 293 cells. Expression was verified by measuring binding of [3H]Ro 15-1788 to membrane fractions. On coexpression with wild-type subunits to yield alpha beta gamma combinations, the mutants alpha T206A and alpha Y209F each resulted in maximal [3H]Ro 15-1788 binding comparable with wild-type receptors, whereas alpha Y209A resulted in ~10% of the binding of wild-type receptors.

In alpha Y209Qbeta gamma receptors, no binding was detectable of [3H]Ro 15-1788 or [3H]flunitrazepam, and the ligand for the agonist site of the receptor [3H]muscimol was used. On coexpression with beta gamma , alpha Y209Q resulted in maximal [3H]muscimol binding approximately half that of wild-type receptors (Fig. 1). Specific binding of [3H]muscimol to alpha Y209Qbeta gamma and wild-type receptors showed a KD value of 14 ± 6 and 14 ± 5 nM, and a maximal binding capacity of 0.74 ± 0.14 and 1.6 ± 0.6 pmol/mg of protein, respectively, showing that at least this mutation, which destroyed the binding of two different radiolabeled ligands of the benzodiazepine binding site, did not significantly affect agonist binding affinity. In nontransfected cells, a very small endogenous component of specific [3H]muscimol binding was seen similar to that previously observed (27), but because of its small size, it could not be properly quantified.


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  		Fig. 1.   Binding of [3H]muscimol to wild-type and alpha Y209Qbeta gamma receptors. HEK 293 cells were transiently transfected with the wild-type or the mutated alpha  subunit, each combined with wild-type beta  and gamma subunits. After harvesting and washing of the membranes, binding assays with [3H]muscimol were carried out as described in Materials and Methods. Wild-type (bullet ) and mutant alpha Y209Qbeta gamma receptors (open circle ) bound the agonist with a Kd value of 6 and 13 nM and a Bmax value of 1.1 and 0.6 pmol/mg of protein, respectively, in this experiment. Values are mean ± standard deviation of two determinations performed in triplicate.

Expression of the alpha 1 subunit was also verified on Western blot analysis (25) for these subunit combinations. Although nontransfected cells did not result in a signal, wild-type and mutant alpha Y209Qbeta gamma receptors resulted in a signal of comparable intensity (not shown).

Surface expression in living HEK 293 cells was followed for wild-type receptors and alpha Y209Abeta gamma and alpha Y209Qbeta gamma mutant receptors. This was achieved using fluorescent staining of GABAA receptors on living cells with an alpha 1-specific polyclonal antibody (23, 24) recognizing an extracellular epitope followed by fluorescence microscopy. Although cells transfected with beta gamma resulted only in very little autofluorescence (Fig. 2b), cells transfected with wild-type receptors showed an intense fluorescence organized in clusters at the surface membrane with little background fluorescence in the cell interior (Fig. 2d). Cells transfected with mutant alpha Y209Qbeta gamma (Fig. 2f) displayed a surface staining similar to wild-type receptors, and alpha Y209Abeta gamma receptors seemed to show slightly less fluorescence (not shown). The organization of the fluorescence into clusters is induced by the polyclonal antibody and lateral diffusion of the receptors during the incubation; a random distribution was found in transfected cells that were first fixed and then immunolabeled (Fig. 2j). Again, cells transfected with beta gamma only did not show any specific fluorescence (Fig. 2h). The results demonstrate clearly that in these experiments, mutant subunits alpha Y209Q reach the surface membrane to a similar extent as the wild-type subunits. Furthermore, these observations made with the alpha Y209Qbeta gamma mutant parallel our observations in [3H]muscimol binding measurements.


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  		Fig. 2.   Surface immunofluorescence of GABAA receptors expressed in HEK 293 cells. GABAA receptors were stained on living and fixed cells, using an antibody recognizing an extracellular epitope as described in Materials and Methods and visualized using a Zeiss Axiovert 35 fluorescence microscope. a, c, e, g, and i, Differential interference contrast (DIC) optics. b, d, f, h, and j, Corresponding immunofluorescence pictures. a, b, g, and h, Cells transfected with beta gamma receptors. c, d, i, and j, Cells transfected with wild-type alpha beta gamma receptors. e and f, Cells transfected with alpha Y209Qbeta gamma receptors. a-f, Living cells. g-j, Fixed cells. Scale bar, 10 µm.

Binding properties of alpha Y209Abeta gamma , alpha Y209Fbeta gamma , and alpha Y209Qbeta gamma receptors. In the previous functional study (15), the mutated amino acid Y209A of the alpha  subunit yielded similar but somewhat differing behavior toward diazepam and zolpidem. Although there was a significant difference between wild-type channels and channels containing this mutation toward 1 µM zolpidem, this difference was not significant for 1 µM diazepam; therefore we investigated whether binding of ligands was affected.

When the wild-type tyrosine was replaced by alanine, affinities for [3H]Ro 15-1788 and diazepam (Fig. 3) were reduced 40-50-fold, whereas zolpidem was unable to displace all specific [3H]Ro 15-1788 binding at concentrations up to 30 µM and had therefore lost the ability to bind to the receptor site (Table 1).


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  		Fig. 3.   Displacement of [3H]Ro 15-1788 binding from alpha Y209Abeta gamma receptors by diazepam. HEK 293 cells were transiently transfected with the wild-type or mutated alpha  subunit, each combined with wild-type beta  and gamma  subunits. After harvesting and washing of the membranes, displacement of 2 and 20 nM [3H]Ro 15-1788 for wild-type (bullet ) and mutant (open circle ) receptors with varying concentrations of diazepam is shown. Values are mean ± standard deviation of two determinations performed in duplicate.

                              
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  		TABLE 1
Binding properties of receptor containing alpha 206 and alpha 209 mutants

Wild-type or mutated GABAA receptors were transiently expressed in HEK 293 cells. Resultant binding properties for ligands of the benzodiazepine binding site were measured. Kd values for the receptors were determined by binding of [3H]Ro 15-1788. Ki values for each compound were determined by displacement of [3H]Ro 15-1788 binding and were calculated according to the equation of Cheng and Prusoff (21). IC50 values for each compound were determined by nonlinear least-squares regression. Data are mean ± standard deviation of two determinations performed in duplicate.

Replacement of tyrosine with phenylalanine (removing a hydroxyl group from residue 209 of the alpha  subunit) led to a small decrease (2-8-fold) for [3H]Ro 15-1788, diazepam, zolpidem, DMCM, and Cl 218872 (Table 1). The largest effect was seen on DMCM (Fig. 4) binding, indicating that this hydroxyl residue is important for interaction of the receptor with this compound.


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  		Fig. 4.   Displacement of [3H]Ro 15-1788 binding from alpha Y209Fbeta gamma receptors by DMCM. HEK 293 cells were transiently transfected with the wild-type or mutated alpha  subunit, each combined with wild-type beta and gamma  subunits. After harvesting and washing of the membranes, displacement of 2.1 and 7.6 nM [3H]Ro 15-1788 for wild-type (bullet ) and mutant (open circle ) receptors with varying concentrations of DMCM is shown. Values are mean ± standard deviation of two determinations performed in duplicate.

When the same amino acid residue tyrosine in position 209 of the alpha  subunit was replaced by glutamine, the binding properties of the benzodiazepine site were altered dramatically. Indeed, we first analyzed for binding of [3H]Ro 15-1788 and were unable to detect any specific binding. Before immunofluorescence, Western blot experiments, and [3H]muscimol binding, we interpreted this as a failure in the expression of the mutant receptor.

When [3H]flunitrazepam was used for binding, a saturable binding with a KD value of 43 ± 6 nM (two experiments) and a maximal specific binding capacity of ~400 fmol/mg of protein could be measured (not shown). Displacement studies with Ro 15-1788, zolpidem, DMCM, and diazepam were then carried out. An affinity of <30 µM was not detectable for Ro 15-1788, zolpidem, DMCM, or Cl 218872, indicating that these ligands were unable to displace [3H]flunitrazepam. These binding properties are reminiscent of the [3H]flunitrazepam binding site endogenous to HEK 293 cells (28). Fuchs et al. (28) reported an endogenous binding component for [3H]flunitrazepam. They found a specific binding of 20 ± 1 fmol/mg of protein at a 2 nM concentration. The Kd value was estimated at >100 nM. If the value for specific binding is extrapolated to 20 nM [3H]flunitrazepam, binding amounts to ~200 fmol/mg of protein. Specific binding also could not be displaced by Ro 15-1788 or a beta -carboline. We found that in the presence of 20 nM [3H]flunitrazepam, ~120 fmol/mg of protein was specifically bound to the membranes, regardless of whether cells were untransfected or transfected with the Y209Q mutant. Flunitrazepam therefore does not bind to expressed receptors but rather to an unidentified endogenous binding site. As shown above, measurement of [3H]muscimol binding showed that the receptor was present and, remarkably, the binding affinity for the receptor agonist muscimol was unaltered. Because [3H]muscimol was bound to alpha Y209Qbeta gamma and the receptor appears on the cell surface, the mutation Y209Q leads to an intact receptor but to the complete loss of binding ability for the radiochemicals [3H]flunitrazepam and [3H]Ro 15-1788.

Functional properties of alpha Y209Qbeta gamma receptors. Wild-type alpha beta gamma and mutant alpha Y209Qbeta gamma receptors were expressed in X. laevis oocytes. The mutant receptor resulted in maximal GABA current amplitudes approximately three times smaller than those of wild-type receptors. The apparent affinities for GABA to activate ion currents (Ka) were determined for both types of receptor. Fig. 5, top, shows that there is a small, nearly 2-fold reduction in Ka value from 27 ± 4 (three experiments) in wild-type to 48 ± 7 µM (three experiments) in mutant receptors. Concentrations of flunitrazepam of <30 nM failed in contrast to wild-type receptors to significantly stimulate GABA-induced currents (Fig. 5, bottom). Higher concentrations of flunitrazepam resulted in a small current stimulation in mutant receptors, amounting to <15% of the wild-type at 1 µM flunitrazepam..


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  		Fig. 5.   Loss of stimulation by low concentrations of flunitrazepam of alpha Y209Qbeta gamma receptors. Wild-type alpha beta gamma (bullet , black-square) and mutant alpha Y209Qbeta gamma (open circle , square ) receptors were functionally expressed in X. laevis oocytes. Top, GABA dose-response curves; increasing concentrations of GABA were applied, and data were treated as indicated in Materials and Methods. Values are mean ± standard deviation of three determinations. Bottom, effect of flunitrazepam; increasing concentrations of flunitrazepam were applied together with 5 µM GABA. The relative stimulation of GABA currents in the absence of flunitrazepam is shown. Values are mean ± standard deviation of four determinations.

Binding properties of alpha T206Abeta gamma receptors. In a previous functional study using receptors expressed in X. laevis oocytes and electrophysiological techniques (15), this mutation resulted in channels displaying an enhanced response to diazepam and zolpidem. It was therefore interesting to see how the binding of ligands of the benzodiazepine binding site was affected. This point mutation led to the expression of receptors displaying a slightly reduced affinity for Ro 15-1788, diazepam, and zolpidem (2-, 4-, and 5-fold) (Table 1). Interestingly, the affinities for DMCM and Cl 218872 were increased strongly, by 5- and 16-fold (Fig. 6), respectively, indicating that these latter ligands can be better accommodated after the replacement of threonine by alanine.


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  		Fig. 6.   Displacement of [3H]Ro 15-1788 binding from alpha T206Abeta gamma receptors by diazepam. HEK 293 cells were transiently transfected with the wild-type or mutated alpha  subunit combined with wild-type beta  and gamma  subunits. After harvesting and washing of the membranes, displacement of 2.1 and 3.0 nM [3H]Ro 15-1788 for wild-type (bullet ) and mutant (open circle ) receptors with varying concentrations of Cl 218872 is shown. The mutant displays clearly higher affinity for Cl 218872 than wild-type receptors. Values are mean ± standard deviation of two determinations performed in duplicate.

    Discussion
Summary
Introduction
Materials & Methods
Results
Discussion
References

We attempted to further characterize the structural and functional properties of the binding site for benzodiazepine-site ligands. For this purpose, wild-type and mutant GABAA receptors were expressed in HEK 293 cells and X. laevis oocytes. Expression was verified by Western blot analysis, immunocytochemistry, radioligand binding, and electrophysiology.

Binding properties of wild-type and mutant alpha Y209Abeta gamma , alpha Y209Fbeta gamma , alpha Y209Qbeta gamma , and alpha T206Abeta gamma receptors. Wild-type and mutant GABAA receptors were expressed by transient transfection in HEK 293 cells. The agonist binding properties and those for allosteric modulators acting at the benzodiazepine binding site were determined using [3H]muscimol and [3H]Ro 15-1788, respectively. Although the first type of binding site was unaltered, at least in the Y209Q mutant, the second type was profoundly affected in its properties. Because the endogenous [3H]flunitrazepam binding site to HEK 293 cells is unable to bind the antagonist Ro 15-1788, in all cases in which experiments were based on [3H]Ro-15 1788 binding (i.e., all reported mutants mentioned in Table 1), this endogenous site can be safely ignored. Both mutant receptors alpha T206Abeta gamma and alpha Y209Fbeta gamma were affected up to 16-fold in their binding properties. Interestingly, replacement of the threonine residue in position 206 by alanine led to an increase in affinity for the negative allosteric modulator DMCM and the partial positive allosteric modulator Cl 218872, amounting to 5- and 16-fold, respectively. It might be speculated that reduction in residue size leads to better accommodation of these ligands in the binding pocket. The mutant receptor alpha Y209Abeta gamma showed reduced affinities of >40-fold for several ligands. Reduction in size and loss of aromaticity of this amino acid residue lead to the retention of some affinity (40-50-fold reduction) for diazepam and Ro 15-1788, whereas any detectable affinity for zolpidem, DMCM, and Cl 218872 is lost.

Replacement of tyrosine in position 209 in the alpha  subunit by the similar-sized, nonaromatic residue glutamine leads to the complete loss of any detectable affinity for the two benzodiazepine ligands tested, [3H]Ro 15-1788 and [3H]flunitrazepam (Table 1). The binding properties of the alpha Y209Qbeta gamma receptor (i.e., normal presence of agonist binding and absence of the benzodiazepine ligand binding site) are reminiscent of dual subunit receptor alpha beta , lacking the gamma  subunit. Because benzodiazepine binding is still present in alanine and phenylalanine mutants of the same residue but each expresses an altered pharmacology, we believe this is an unlikely interpretation of our results. Rather, we believe the binding site for these ligands is compromised by the presence of the glutamine residue. Also, after expression in X. laevis oocytes, the alanine mutant still results in diazepam stimulation (15), which indicates the presence here of a gamma  subunit. In addition, we have shown using immunocytochemical methods that the altered alpha  subunit reaches the surface membrane in living cells. Because the exclusive expression of alpha  subunits results in the very inefficient formation of homomeric channels (8, 29, 30), the mutant alpha  subunit is probably assembled together with other subunits.

Relation between binding studies and receptor function. Functional studies on receptors expressed in X. laevis oocytes were previously been performed with the alanine mutants alpha T206Abeta gamma and alpha Y209Abeta gamma (15). The first mutant channel was stimulated by diazepam and zolpidem to a significantly higher degree than the wild-type channel. For the second mutant, diazepam resulted in a nonsignificantly reduced stimulation, and zolpidem resulted in a loss of stimulation. For functional effects to occur, a substance must first bind. The fact that diazepam and zolpidem result in differential decreases in stimulation of the currents elicited in alpha Y209Abeta gamma can also be rationalized on the basis of the binding data, showing an affinity of ~0.5 µM for diazepam and a very strongly reduced affinity for zolpidem. Thus, at the 1 µM concentration used, these agents are expected to occupy their receptor site to a degree of 67% and <4%, respectively.

The glutamine mutant alpha Y209Qbeta gamma showed a <2-fold reduction in the apparent affinity for channel gating by GABA (Fig. 5, top), whereas any stimulation of the GABA-induced currents by flunitrazepam below a concentration of 30 nM was absent (Fig. 5, bottom). This confirms our findings with the immunocytochemistry and binding studies of this receptor variant showing that the mutant receptor reaches the surface membrane and findings for binding properties in transiently transfected 293 cells, with the binding site for [3H]muscimol unaltered and binding sites for [3H]Ro 15-1788 and [3H]flunitrazepam undetectable.

Spacing of mutations affecting the binding properties of ligands of the benzodiazepine binding site. The mutations described here affect the two amino acid residues T206 and Y209 of the alpha  subunit. These residues are located three amino acids apart, which suggests that this region of the alpha  subunit might form an alpha  helix. A Chou-Fasman analysis (31) predicts for region 199-209 an alpha  helix followed by a beta  sheet. Amino acid 206 is located in the center of a hydrophilic region, and 209 is located at the interface of the latter and a hydrophobic region. If the prediction of the secondary structure is correct for this stretch of the alpha  subunit and if amino acids altering the binding properties of ligands of the benzodiazepine binding site do take part in the formation of the actual binding pocket, constraints are imposed on the way in which the pocket is formed by the protein.

Homology of alpha 206 and alpha 209 with beta 202 and beta 205. The two residues described here that extensively affect binding of allosteric modulators, Thr206 and Tyr209 on the alpha 1 subunit, are directly homologous to Thr202 and Tyr205 on the beta 2 subunit, as implicated in the interaction with the channel agonist GABA (9). This indicates a large degree of homology between the binding sites for channel agonists and for channel modulators of the benzodiazepine type. During revision of the current report, a study by Amin et al. (32), in which the authors made a similar conclusion, came to our attention. Based on functional studies in X. laevis oocytes and on binding studies of the serine mutant of the tyrosine alpha 209, it was concluded, similar to the current report, that this amino acid residue may be involved in binding of benzodiazepines.

Structure of the binding site. The following amino acid residues on the alpha 1 subunit, or homologous residues on other alpha  isoforms, and on the gamma 2 subunit have been shown to affect functional alterations to the response of ligands of the benzodiazepine binding site: alpha Y159, alpha Y161, alpha T206, alpha Y209, gamma F77, gamma M130, and gamma T142 (14-17, 32, current report). The binding of these ligands is affected by alpha H101, alpha Y159, alpha G200, alpha T206, alpha Y209, gamma F77, and gamma M130 (16, 17, 32-35; current report). These seven residues have been shown to strongly affect the binding properties. Unless these amino acid residues exert distal effects, they might take part in the formation of the binding pocket for ligands of the benzodiazepine binding site. These amino acid residues are located in five different areas of the protein complex, which may be located next to each other. This means that both the alpha subunit and the gamma  subunit may contribute to the formation of the binding site, each folding back after > 50 residues. The proper three-dimensional arrangement of these residues remains to be established.

    Acknowledgments

We are grateful to Prof. H. Reuter, in whose institute this work was carried out, for continuous encouragement; to Prof. W. Sieghart (University of Vienna, Austria) for providing the alpha 1 subunit-specific antibody in the context of the collaboration funded by the European Union; to Dr. K. Kannenberg for expert help with microscopic techniques; and to Dr. V. Niggli for careful reading of the manuscript.

    Footnotes

Received April 21, 1997; Accepted June 20, 1997

   This work was supported by Grant 31-37192.93 from the Swiss National Science Foundation, EU Grant BIO4-CT96-0585 (BWW 96.0010), and the Foundation for the Promotion of Scientific Research at the University of Bern.

Send reprint requests to: Dr. Erwin Sigel, Department of Pharmacology, University of Bern, Friedbühlstr. 49, CH-3010 Bern, Switzerland. E-mail: sigel{at}pki.unibe.ch

    Abbreviations

GABA, gamma -aminobutyric acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMCM, methyl-6,7-dimethoxy-4-ethyl-beta -carboline-3-carboxylate; HEK, human embryonic kidney.

    References
Summary
Introduction
Materials & Methods
Results
Discussion
References

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Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics


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