Flunitrazepam Has an Inverse Agonistic Effect on Recombinant alpha 6beta 2gamma 2-GABAA Receptors via a Flunitrazepam-binding Site*

(Received for publication, January 21, 1997)

Charlotte A. E. Hauser Dagger , Christian H. R. Wetzel §, Barbara Berning , Franz M. Gerner and Rainer Rupprecht

From the Max Planck Institute of Psychiatry, Clinical Institute, Kraepelinstrasse 10, 80804 Munich, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

gamma -Aminobutyric acid type A (GABAA) receptor subtypes containing the alpha 6-subunit are generally thought to be insensitive to the action of benzodiazepine agonists. We describe the specific binding of the benzodiazepine agonist flunitrazepam to alpha 6beta 2gamma 2-containing GABAA receptors, which has not been observed before and differs from previous reports. With the whole-cell voltage-clamp technique, we observed a functional discrimination between alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors. Different benzodiazepines had different effects on GABA-evoked chloride currents. The agonist flunitrazepam had an inverse agonistic effect, whereas the antagonist flumazenil increased GABA-induced chloride currents. The action of flunitrazepam on the channel activity of alpha 6beta 2gamma 2-receptors was opposite to its action on alpha 1beta 2gamma 2-receptors. We conclude that flunitrazepam can act as either an agonist or an inverse agonist, depending on the GABAA receptor configuration.


INTRODUCTION

Benzodiazepines (BZs)1 act via the gamma -aminobutyric acid type A (GABAA) receptor, thereby influencing the chloride influx into the cell (1, 2). Studies of a variety of GABAA receptor compositions have led to the conclusion that ternary receptors reconstituted from alpha -, beta -, and gamma -subunits have binding sites for BZs, with the high-affinity site being located on the alpha -subunit (3, 4). These subunits are differentially distributed throughout the central nervous system (5, 6). It is generally thought that the subunit composition of the GABAA receptor complex organizes the receptor's pharmacology. The best known concept is that the alpha 1beta xgamma 2-receptors, called type I BZ receptors, preferentially bind full BZ agonists with high affinity (3) and that the alpha 2/3/5beta xgamma 2-receptors, classified as type II BZ receptors, have a 10-fold lower affinity for BZ agonists (7-10). The view is widely held that alpha 6- and alpha 4-containing receptors have virtually no affinity for full BZ agonists (11-13), although they retain a high affinity for the partial inverse BZ agonist Ro 15-4513 (14, 15). In the mature mammalian brain, the alpha 6-subunit is expressed exclusively in the cerebellum (6), where it is preferentially found in granular cells (16).

The studies we report here focus on the BZ pharmacology of the alpha 6beta 2gamma 2-receptor. We show that alpha 6beta 2gamma 2-GABAA receptors have high affinity for the BZ agonist flunitrazepam, although the histidine at position 100 in the ligand-binding domain, which is thought to be crucial for BZ binding, is missing (13). Furthermore, using the whole-cell voltage-clamp technique on alpha 6beta 2gamma 2-transfected cells, we demonstrate that flunitrazepam has an inverse agonistic effect on GABA-induced chloride currents, whereas the BZ antagonist Ro 15-1788 (flumazenil) increases GABA-mediated chloride currents.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

Human embryonic kidney cells (HEK 293) were grown and transfected as described (17). In a modification of that method, cells were cotransfected with different rat alpha -variants (alpha 1, alpha 1(Arg-101), alpha 6, and alpha 6(His-100)) in combination with beta 2- and gamma 2-cDNAs (11, 13, 18), using an electroporation system (Biotechnologies & Experimental Research Inc., San Diego, CA) after determination of the optimal electric field strength (19). For optimal receptor expression, final concentrations were as follows: alpha 1, alpha 1(Arg-101), alpha 6, and alpha 6(His-100), 3.3 µg of vector DNA/10-cm tissue culture dish; beta 2, 16.6 µg; and gamma 2, 0.3 µg.

Ligand Binding Assays on Transfected Cells

HEK 293 cells were prepared as described previously (20). For saturation binding analysis with [3H]flunitrazepam or [3H]Ro 15-4513, concentrations between 1.2 and 40 nM were used. Binding experiments were performed in plastic microtiter plates using ~106 cells/well in a total volume of 0.25 ml. The membranes were incubated with the radioactive ligand at 4 °C for 1 h with continuous shaking. In competition experiments, the competing reagent was coapplied with the radioactive ligand. The binding reaction was terminated by rapid filtration through Whatman GF/C filters with a Titertek cell harvester (Skatron Instruments, Newmarket, United Kingdom). The filters were washed twice with 5 ml of ice-cold incubation buffer. Filter-retained radioactivity was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of either 50 µM diazepam or 50 µM Ro 15-4513. Binding assays were analyzed as described (17).

Electrophysiological Recordings

HEK 293 cells were recorded in the whole-cell voltage-clamp configuration of the giga-seal technique (21) under visual control using an inverted microscope (Zeiss, Jena, Germany). The cells were kept in a bath solution containing 150 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgSO4, 10 mM glucose, 20 mM sucrose, and 10 mM HEPES. The pH was adjusted to 7.4 with NaOH. Patch electrodes were pulled from borosilicate glass (Hilgenberg, Malsfeld, Germany) using a horizontal pipette puller (Zeitz Instruments, Augsburg, Germany) to yield pipettes with a resistance of 3-6 megaohms. Pipettes were filled with a solution containing 130 mM KCl, 15 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 2 mM ATP, 0.25 mM cAMP, 5 mM glucose, 10 mM HEPES, and 11 mM EGTA. The pH was adjusted to 7.25 with KOH.

After the whole-cell configuration had been established, the cells were lifted from the glass substrate, and GABA or BZs were applied at the indicated concentrations using a fast superfusion device. A piezo-translator-driven double-barreled application pipette was used to expose the lifted cell to either GABA-free or GABA-containing solution for control experiments (flow rate of 200 µl/min). A 2.5-s GABA pulse was delivered every 60 s. BZs were present at the indicated concentrations in both GABA-free and GABA-containing solutions so that their modulatory properties could be studied. Flunitrazepam and Ro 15-4513 were dissolved in ethanol and diluted with bath solution to the desired concentration. The ethanol concentration was 0.1% in all solutions to avoid any confounding solvent effects. Recordings were obtained from 6 to 12 independent experiments.

Current signals were recorded at a holding potential of -50 mV with an EPC-9 amplifier (Heka, Lambrecht, Germany) using Pulse software on a Macintosh II computer. The data were analyzed using PulseFit (Heka) and IgorPro (Wacemetrics, Lake Oswego, OR) software.


RESULTS AND DISCUSSION

Classical BZ agonists are generally thought to bind to all ternary GABAA receptor complexes reconstituted from alpha -, beta -, and gamma -subunits, except when the alpha 6- or alpha 4-subunit is involved (13). This conclusion was drawn indirectly from competition studies with classical BZ agonists as competitors for the inverse agonist-binding site of [3H]Ro 15-4513. A single histidine was found to be essential for prototypic BZ agonist binding (13). We analyzed the direct binding of [3H]flunitrazepam to recombinant alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-GABAA receptors. The results for the recombinant alpha 6beta 2gamma 2-receptor were unexpected. Scatchard analysis with [3H]flunitrazepam in concentrations between 1.2 and 40 nM revealed a single category of saturable high-affinity binding sites, with a Kd value of 8.7 nM for alpha 6beta 2gamma 2-receptors compared with a Kd value of 3.3 nM for alpha 1beta 2gamma 2-receptors (Fig. 1, A and B). With this finding for alpha 6beta 2gamma 2-receptors, we postulate a binding site for flunitrazepam on this receptor subtype, which has not been detected previously. Whereas alpha 1beta 2gamma 2-receptors are found throughout the central nervous system, alpha 6beta 2gamma 2-receptors are localized predominantly on cerebellar granule cells in the neighborhood of a variety of excitatory ion channels (12, 16).


Fig. 1. Affinity of [3H[]flunitrazepam for regular and mutated alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-GABAA receptors. A, schematic representation of the sequences encoding the rat alpha 1- and alpha 6-subunits and two mutant subunits, in which histidine 101 is replaced by arginine in the alpha 1-subunit and arginine 100 is replaced by histidine in the alpha 6-subunit, as indicated (13). Sequences of the alpha 1-subunit are unshaded, and those of the alpha 6-subunit are shaded. Solid boxes represent the sequence encoding putative membrane-spanning areas. The disulfide bridge is characterized by two lower-case letters c. aa, amino acids. B, affinity of [3H]flunitrazepam for regular and mutated alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors. The numbers indicate the mean Kd values in nM ± S.E. derived from at least three experiments performed in triplicate.
[View Larger Version of this Image (11K GIF file)]

In competition binding studies, the alpha 1beta 2gamma 2-receptors, which are the "classical" GABAA receptors, exhibited the expected displacement of flunitrazepam binding by other BZ agonists (11), whereas for the alpha 6beta 2gamma 2-receptors, we obtained Ki values 150-1000-fold higher than the observed Kd (Table I). The weak displacement of flunitrazepam binding by classical BZ agonists suggested that we had found a binding site on the alpha 6beta 2gamma 2-receptor with a preference for flunitrazepam. We then looked at whether the peripheral BZ receptor ligand 4'-chlorodiazepam (Ro 5-4864), which interacts with a unique low-affinity site on the GABAA receptor, and a peripheral BZ receptor antagonist, the isoquinoline carboxamide derivative PK 11195 (22), bound to the flunitrazepam-binding site. We did not detect any flunitrazepam displacement (Table I). Finally, we examined whether other BZ agonists, such as alprazolam and chlordiazepoxide, or BZ inverse agonists, such as methyl-6,7-dimethoxy-4-ethyl-beta -carboline 3-carboxylate, n-butyl-beta -carboline 3-carboxylate, and desmethyldiazepam, were able to compete for the flunitrazepam-binding site on the alpha 6-subunit, but found only weak displacement or none at all (Table I).

Table I. Binding affinity of various benzodiazepines

Values represent the affinity (Kd or Ki in nM) of alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors for [3H]flunitrazepam and various other benzodiazepines as competitors. Values represent the affinity (Kd or Ki in nM) of alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors for [3H]flunitrazepam and various other benzodiazepines as competitors.
 alpha 1beta 2gamma 2  alpha 6beta 2gamma 2

Kd (nM)a
  [3H]Flunitrazepam 3.3  ± 1.9 8.7  ± 3.0
Ki (nM)
  Diazepam 5.5  ± 2.9 1190  ± 160
  Clonazepam 2.0  ± 0.5 5000  ± 1900
  Bromazepam 190  ± 70 8300  ± 2400
  Lorazepam 5.6  ± 1.2 2900  ± 900
  Ro 5-4864  NDb >10,000
  PK 11195 ND >10,000
  DMCM 8.6  ± 1.8 >10,000
  beta -CCB 28.8  ± 3.0 1580  ± 480
  Chlordiazepoxide 2080  ± 750 >10,000
  Alprazolam 23.1  ± 2.5 >10,000
  Desmethyldiazepam 159  ± 74 2130  ± 700

a Values represent the mean Kd or mean Ki ± S.E. from at least three independent experiments done in triplicate.
b ND, not determined; DMCM, methyl-6,7-dimethoxy-4-ethyl-beta -carboline 3-carboxylate; beta -CCB, n-butyl-beta -carboline 3-carboxylate.

Table II. Binding affinity of [3H]Ro 15-4513 and displacement by various benzodiazepines

Values represent the affinity (Kd or Ki) of a partial inverse benzodiazepine ([3H]Ro 15-4513), a nonselective benzodiazepine antagonist (flumazenil (Ro 15-1788)), and two selective benzodiazepines (flunitrazepam and diazepam) for receptors assembled from a wild-type or mutant subunit (shown) along with a beta 2- and gamma 2-subunit. Values represent the affinity (Kd or Ki) of a partial inverse benzodiazepine ([3H]Ro 15-4513), a nonselective benzodiazepine antagonist (flumazenil (Ro 15-1788)), and two selective benzodiazepines (flunitrazepam and diazepam) for receptors assembled from a wild-type or mutant subunit (shown) along with a beta 2- and gamma 2-subunit.
Receptor type Kd, [3H]Ro 15-4513a Ki
Flunitrazepam Diazepam Flumazenil

nM nM
 alpha 1 19.9  ± 2.0 0.2  ± 0.05 23.4  ± 7.4 10.4  ± 0.5
 alpha 1(Arg-101) 6.6  ± 0.4 >10,000 >10,000 273  ± 15
 alpha 6 6.2  ± 1.9 >10,000 >10,000 149  ± 59
 alpha 6(His-100) 5.1  ± 1.9 1.2  ± 0.01 153  ± 90 96  ± 44

a Values represent the mean Kd or mean Ki ± S.E. from at least three independent experiments done in triplicate.

We also wanted to test whether the Ro 15-4513 binding site on alpha 6beta 2gamma 2 receptors had any influence on the newly described flunitrazepam binding site. Analysis of alpha 1(Arg-101)beta 2gamma 2 and alpha 6(His-100)beta 2gamma 2 receptors along with the wild-type receptors showed that the sensitivity of [3H]Ro 15-4513 binding to flunitrazepam as well as to diazepam coincided with the presence of the crucial amino acid histidine (Table II). The newly proposed high affinity binding site of flunitrazepam on alpha 6beta 2gamma 2 receptors did not compete with the Ro 15-4513 binding site. The BZ antagonist flumazenil exhibited a 100-300-fold lower affinity for mutant receptors and alpha 6beta 2gamma 2 receptors than for alpha 1beta 2gamma 2 receptors which is in agreement with data previously published (13).

To investigate whether an exchange of the crucial amino acid histidine, responsible for high-affinity BZ agonist binding, would change the flunitrazepam affinity for alpha 6beta 2gamma 2-receptors, we used two mutants, already described elsewhere (13), for [3H]flunitrazepam binding studies. These mutant receptors, alpha 1(Arg-101)beta 2gamma 2 and alpha 6(His-100)beta 2gamma 2, showed a single class of binding sites, with mean Kd values of 19.7 and 5.4 nM, respectively (Fig. 1, A and B). This gives evidence that histidine 100 is not the exclusive determinant of flunitrazepam binding as postulated earlier (13).

The response to GABA characterized by the electrophysiological investigation of recombinant GABAA receptors is known to be enhanced consistently by flunitrazepam (23, 24), and this effect can be blocked completely by flumazenil (3, 24). In the case of the rat alpha 1beta 2gamma 2-receptor, 1 µM flunitrazepam has been shown to enhance 1 µM GABA-evoked currents by 64% (25). We observed that alpha 1beta 2gamma 2-receptors reacted as expected to the coapplication of GABA and flunitrazepam. Flunitrazepam (10 µM) markedly affected the amplitude of the peak current at both GABA concentrations used and in a dose-dependent manner. At 1 µM GABA, we detected a 117 ± 46% increase in the amplitude, whereas coapplication with a higher concentration of GABA (10 µM) elicited a smaller potentiation of 46 ± 6% (Fig. 2A).


Fig. 2. Effects of flunitrazepam on recombinant GABAA receptors. Inward currents of HEK 293 cells activated by 1 or 10 µM GABA and measured at -50 mV are shown for alpha 1beta 2gamma 2-receptors (A) and alpha 6beta 2gamma 2-receptors (B) as a representative experiment of 6-12 independent experiments. Transfected cells were first challenged with GABA for 5 s and again challenged with GABA plus 10 µM flunitrazepam. Black curves (b) are control GABA responses for the situation using either 1 µM GABA (left panels) or 10 µM GABA (right panels) without flunitrazepam. Gray curves (a) are GABA responses in the presence of 10 µM flunitrazepam. Also shown (C) is the effect of 10 µM flunitrazepam on alpha 6beta 2gamma 2-receptors at different concentrations of GABA expressed as mean ± S.E. Data are expressed in terms of percent inhibition, which was calculated by the following formula: (1 - IGABA+10 µM flunitrazepam/IGABA) × 100. The asterisks indicate a significant inhibitory effect (t test for paired samples; p < 0.05).
[View Larger Version of this Image (23K GIF file)]

For alpha 6beta 2gamma 2-receptors, however, 10 µM flunitrazepam and 10 µM GABA resulted in a significant reduction of control currents to 76 ± 12%, whereas coapplication of 1 µM GABA and 10 µM flunitrazepam did not produce a significant change in amplitude (Fig. 2, B and C). This effect of flunitrazepam was shown to be dependent on the concentration of GABA and became significant at a concentration of 6 µM GABA (Fig. 2C). Our data thus indicate that flunitrazepam acts on alpha 6beta 2gamma 2-receptors in an inverse agonistic manner.

Our investigation of the effects of flumazenil on GABA-evoked chloride currents showed opposite effects for the two receptors studied. For cells expressing alpha 1beta 2gamma 2-receptors, 10 µM flumazenil reduced the amplitude of chloride currents induced by application of 10 µM GABA to 89 ± 3% of control, whereas for cells expressing alpha 6beta 2gamma 2-receptors, there was an increase to 118 ± 3% of control induced by application of 1 µM GABA and 10 µM flumazenil (Figs. 3 (A and B) and 4A). We have used different GABA concentrations depending on the receptor subtype because we found in an earlier investigation (17) that alpha 6beta 2gamma 2-receptors effectively responded to GABA at lower concentrations than alpha 1beta 2gamma 2-receptors. The effect of flumazenil on alpha 6beta 2gamma 2-receptor-mediated responses is low compared with previous reports on Sf-9 cells and HEK 293 cells (26). In an earlier investigation, an apparent agonistic activity of flumazenil was reported for a binary GABAA receptor using the rat alpha 1- and beta 2-subunits expressed in Xenopus oocytes (23). Here, 10 µM flumazenil potentiated the response induced by 2-10 µM GABA to 175% of control.


Fig. 3. Effects of flumazenil or Ro 15-4513 on recombinant GABAA receptors. Inward currents of HEK 293 cells activated by 1 µM (B) or 10 µM (A) GABA and measured at -50 mV are shown for alpha 1beta 2gamma 2-receptors (A) and alpha 6beta 2gamma 2-receptors (B) as a representative experiment of 6-12 independent experiments. Transfected cells were similarly treated and recorded as described in the legend of Fig. 2. Black curves (b) are control GABA responses for either 1 µM (B) or 10 µM (A) GABA without BZ. Gray curves (a) are GABA responses in the presence of either flumazenil (left panels) or Ro 15-4513 (right panels). For flumazenil (10 µM) application, in cells expressing alpha 6beta 2gamma 2-receptors, application of 1 µM GABA induced chloride currents with a mean amplitude of 639 ± 213 pA, whereas in cells expressing alpha 1beta 2gamma 2-receptors, application of 10 µM GABA induced chloride currents with a mean amplitude of 2585 ± 435 pA. For Ro 15-4513 (1 µM) application, in cells expressing alpha 6beta 2gamma 2-receptors, application of 1 µM GABA induced chloride currents with a mean amplitude of 984 ± 230 pA, whereas in cells expressing alpha 1beta 2gamma 2-receptors, application of 10 µM GABA induced chloride currents with a mean amplitude of 2835 ± 655 pA.
[View Larger Version of this Image (29K GIF file)]


Fig. 4. Relative change in the amplitude of GABA-induced chloride currents of recombinant alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors. A, application of different BZ receptor ligands to alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors; B, coapplication of flunitrazepam and flumazenil. HEK 293 cells expressing alpha 1beta 2gamma 2- or alpha 6beta 2gamma 2-receptors were first challenged with GABA for 5 s. Cells were again challenged with GABA plus 10 µM flunitrazepam. A washout period of 5 min followed. Cells were further challenged with GABA plus 10 µM flunitrazepam and 10 µM flumazenil. After a new 5-min washout period, the last application of GABA plus 10 µM flumazenil followed. Transfected cells were treated similarly as described in the legend of Fig. 3 using 1 µM GABA for alpha 6beta 2gamma 2-receptors and 10 µM GABA for alpha 1beta 2gamma 2-receptors.
[View Larger Version of this Image (28K GIF file)]

Coapplication of flunitrazepam and flumazenil to recombinant alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors (Fig. 4B) showed that in the case of the alpha 6beta 2gamma 2-receptors, the inverse agonistic properties of flunitrazepam were reduced by the agonistic action of flumazenil. As expected, for the alpha 1beta 2gamma 2-receptors, the agonistic properties of flunitrazepam were almost completely suppressed by flumazenil.

The partial inverse agonist Ro 15-4513 had the opposite effect on GABA-induced currents in alpha 1beta 2gamma 2- and alpha 6beta 2gamma 2-receptors. In cells expressing alpha 1beta 2gamma 2-receptors, 1 µM Ro 15-4513 decreased the amplitude of 10 µM GABA-induced chloride currents. For alpha 6beta 2gamma 2-receptors with 1 µM GABA, there was a comparable change in current amplitude, but in the opposite direction (Figs. 3 (A and B) and 4A).

In this study, we demonstrate that the two receptor subtypes alpha 1beta 2gamma 2 and alpha 6beta 2gamma 2 react with the BZ receptor ligands flunitrazepam, flumazenil, and Ro 15-4513 in a functionally opposite manner. The exact mechanism underlying the distinct BZ pharmacology of the alpha 6beta 2gamma 2-receptor is not clear at present. Our findings, together with those of earlier investigations (13, 26), lead us to conclude that distinct amino acids of the BZ-binding pocket determine the BZ agonistic or inverse agonistic activity of the GABAA receptor subtype. For the wild-type alpha 6beta 2gamma 2-receptor, we postulate that after binding of flunitrazepam, the bulky amino acid arginine induces a structural change that modifies the channel in a way that decreases chloride ion flux. If the smaller amino acid histidine (wild-type alpha 1beta 2gamma 2-receptor) is present, such a hindrance of influx may be absent. Studies involving site-directed mutagenesis should be conducted to elucidate the molecular characteristics of the flunitrazepam-binding site on the alpha 6beta 2gamma 2-receptor.


FOOTNOTES

*   This work was supported by a grant from the Gerhard-Hess-Programm of the Deutsche Forschungsgemeinschaft (to R. R.).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.
Dagger    To whom correspondence should be addressed. Tel.: 49-89-30622-626; Fax: 49-89-30622-605; E-mail: chhauser{at}mpipsykl.mpg.de.
§   Present address: Dept. of Cell Physiology, Ruhr University Bochum, 44780 Bochum, Germany.
1   The abbreviations used are: BZs, benzodiazepines; GABA, gamma -aminobutyric acid; GABAA, gamma -aminobutyric acid type A.

ACKNOWLEDGEMENTS

We are grateful to Peter Seeburg and Heike Wieland for providing the GABAA receptor cDNAs, Hartmut Lüddens for supplying several BZs, and Thorsten Trapp for critical comments on the manuscript. We are also grateful to Thomas Kuckuk and Bettina Burkart-Lauer for excellent assistance with the artwork.


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