High-affinity Binding Sites For Benzodiazepines Research Articles

Peripheral gating of mechanosensation by glial diazepam binding inhibitor.

We report that diazepam binding inhibitor (DBI) is a glial messenger mediating crosstalk between satellite glial cells (SGCs) and sensory neurons in the dorsal root ganglion (DRG). DBI is highly expressed in SGCs of mice, rats, and humans, but not in sensory neurons or most other DRG-resident cells. Knockdown of DBI results in a robust mechanical hypersensitivity without major effects on other sensory modalities. In vivo overexpression of DBI in SGCs reduces sensitivity to mechanical stimulation and alleviates mechanical allodynia in neuropathic and inflammatory pain models. We further show that DBI acts as an unconventional agonist and positive allosteric modulator at the neuronal GABAA receptors, particularly strongly affecting those with a high-affinity benzodiazepine binding site. Such receptors are selectively expressed by a subpopulation of mechanosensitive DRG neurons, and these are also more enwrapped with DBI-expressing glia, as compared with other DRG neurons, suggesting a mechanism for a specific effect of DBI on mechanosensation. These findings identified a communication mechanism between peripheral neurons and SGCs. This communication modulates pain signaling and can be targeted therapeutically.

Positive allosteric modulation of native and recombinant GABAA receptors by hops prenylflavonoids

Hops are a major component of beer that is added during brewing. In addition to its wide range of bioactivity, it exhibits neuroactive properties as a sedative and sleeping aid. The compounds responsible for this activity are yet to be revealed and understood in terms of their pharmacological properties. Here we evaluated the potential of several hops flavonoids in modulating the GABAergic activity and assessed their selectivity to GABAA receptors subtypes. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native and recombinant α1β3γ2, α2β3γ2 and α6β3δ receptors expressed in HEK293 cells. Flumazenil sensitivity of GABA-potentiating effects and [3H]Ro 15-4513 binding assay were used to examine the flavonoids binding to benzodiazepine site. The prenylflavonoids xanthohumol (XN), isoxanthohumol (IXN) and 8-prenylnaringenin (8PN) potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner. The IC50 for this potentiation in native GABAA receptors were 29.7 µM, 11.6 µM, 7.3 µM, respectively. In recombinant receptors, the sensitivity to prenylflavonoid potentiation of GABA-induced displacement of [3H]EBOB binding followed the order α6β3δ > α2β3γ2 > α1β3γ2 with the strongest inhibition observed by 8PN in α6β3δ (IC50 = 3.6 μM). Flumazenil had no significant effect on the prenylflavonoid-induced displacement of [3H]EBOB binding and [3H]Ro 15-4513 displacement from native GABAA receptors was only detected at high micromolar concentrations (100 µM). We identified potent prenylflavonoids in hops that positively modulate GABA-induced responses in native and αβγ/δ recombinant GABAA receptors at low micromolar concentrations. These GABAergic modulatory effects were not mediated via the high-affinity benzodiazepine binding site.

Antidepressant, Anxiolytic and Antinociceptive Activities of Constituents from Rosmarinus Officinalis.

Rosmarinus officinalis, traditionally known as rosemary, has been widely used in traditional medicines and has long been known as the herb of remembrance. However, few studies have investigated the effects of non-volatile components of rosemary on central nervous system function. Fractionation of R. officinalis led to the isolation of salvigenin, rosmanol and cirsimaritin, which were investigated in mouse models of acute toxicity, antinociception (tail immersion and hot plate tests), depression (tail suspension and forced swim tests) and anxiety (elevated plus maze and light/dark box paradigms). Rosmanol, cirsimaritin and salvigenin were not found to exhibit any signs of acute toxicity (50-200 mg/kg), but elicited antinociceptive, antidepressant and anxiolytic activities. Rosmanol, cirsimaritin and salvigenin, all previously shown to have biphasic modulation of GABAA receptors, demonstrated CNS activity in mouse models of antinociception, antidepressant and anxiolysis. The anxiolytic activity of all three compounds was not ameliorated by flumazenil, but was inhibited by pentylenetetrazol, suggesting a mode of action via GABAA receptors at a site other than the high affinity benzodiazepine binding site. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ− subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ− site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Comparing the high affinity benzodiazepine binding site with the homologous “CGS 9895” site in GABA‐A receptors (1059.1)

GABA‐A receptors are the major inhibitory transmitter receptors in the brain and the site of action of many clinically important drugs. CGS 9895 and many other pyrazoloquinolinones are high affinity null modulators at the benzodiazepine (Bz) binding site and at higher concentrations also modulate the receptors via the α+β‐ interface. To gain insights into the interactions of CGS 9895 with both binding sites, we used our homology models to select γ2‐ side amino acid residues important for Bz binding and to incorporate them into the β3‐ side by site directed mutagenesis. These mutated β3 subunits were co‐expressed with α subunits. The recombinant receptors were investigated by the two electrode voltage clamp method in Xenopus laevis oocytes and by radioligand binding assays. With only four mutations we were able to introduce a high affinity binding site for CGS 9895 into αβ3 receptors. Furthermore, GABA currents of several recombinant αβ3(mut) receptors could be modulated by the tested pyrazoloquinolinones and by other ligands of the high affinity Bz binding site, but not by diazepam. We thus introduced a subsite of the high affinity Bz binding site into the α+β3‐ interface.Grant Funding Source: Supported by FWF W1232 Molecular Drug Targets

Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β− interface

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2-(4-methoxyphenyl)-2H-pyrazolo[4,3-c]quinolin-3(5H)-one) acts as a null modulator (antagonist) at the high affinity benzodiazepine binding site, but in addition elicits a strong enhancement of GABA-induced currents via a novel drug binding site at the extracellular α+β- interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the α1+β3- interface of GABAA receptors. GABAA receptors were expressed in Xenopus laevis oocytes and investigated by the two-electrode voltage clamp method. We not only identified compounds with higher efficacy/potency than CGS 9895 for stimulating GABA-induced currents via the α1+β3-binding site, but also discovered compounds acting as null modulators at this site. Most of the compounds also acted as null modulators via the benzodiazepine binding site of GABAA receptors. But some of the positive allosteric modulators or null modulators exclusively exerted their action via the α+β- binding site. Pyrazoloquinolinones and pyrazolopyridinones represent the first prototype of drug candidates mediating benzodiazepine like modulatory effects via the α+β-interface of GABAA receptors. The discovery of null modulators acting as inhibitors of the plus modulators provides a highly useful tool for the discovery of additional classes of compounds that can modulate GABAA receptors via this site, which may lead to novel therapeutic principles.

A conserved Asparagine Between Loops A and E of Anion Ligand-Gated Channels is Critical for Gaba(A) Receptor Function

Cys-loop ligand-gated ion channels are pentameric transmembrane proteins that are essential for the regulation of neuronal excitability. In synaptic GABA(A) receptors, residues in the two α-β+ interfaces are known to define the GABA binding site while homologous residues in the γ-α+ interface define the classical high-affinity benzodiazepine binding site. Very little however, is known about the structure and function of the remaining two interfaces: β-α+ and β-γ+. Since the β- interface is common to both, we chose to investigate the role this interface plays in receptor function, using the tools of structure-homology modeling, site-directed mutagenesis, transient expression of receptors in HEK-293 cells and whole-cell patch-clamp electrophysiology. In this study, we focused on a non-canonical loop domain (NCLD) that links the canonical loops A and E, also known as the β-strand 5-β- strand 5’ linker. Homology models predict that the N-terminal end of this loop flanks a large inter-subunit lumen-accessible crevice. An alanine screen of the NCLD revealed 4 positions within the domain that are critical for receptor activation. Of particular interest was a highly-conserved asparagine: β2(N113). Our data indicate that mutations at this position results in reduced receptor sensitivity to GABA (increased EC50), impaired receptor gating (reduced partial agonist efficacy) and perturbed ion flow (increased rectification). Interestingly, the change in rectification was reversed by the addition of positive allosteric modulators such as propofol and pregnanolone. Taken together, our observations demonstrate the importance of the non-binding interfaces in controlling receptor function.

A Closer Look at the High Affinity Benzodiazepine Binding Site on GABAA Receptors

Ligands of the benzodiazepine binding site of the GABA(A) receptor come in three flavors: positive allosteric modulators, negative allosteric modulators and antagonists all of which can bind with high affinity. The GABA(A) receptor is a pentameric protein which forms a chloride selective ion channel and ligands of the benzodiazepine binding site stabilize three different conformations of this protein. Classical benzodiazepines exert a positive allosteric effect by increasing the apparent affinity of channel opening by the agonist γ-aminobutyric acid (GABA). We concentrate here on the major adult isoform, the α(1)β(2)γ(2) GABA(A) receptor. The classical binding pocket for benzodiazepines is located in a subunit cleft between α(1) and γ(2) subunits in a position homologous to the agonist binding site for GABA that is located between β(2) and α(1) subunits. We review here approaches to this picture. In particular, point mutations were performed in combination with subsequent analysis of the expressed mutant proteins using either electrophysiological techniques or radioactive ligand binding assays. The predictive power of these methods is assessed by comparing the results with the predictions that can be made on the basis of the recently published crystal structure of the acetylcholine binding protein that shows homology to the N-terminal, extracellular domain of the GABA(A) receptor. In addition, we review an approach to the question of how the benzodiazepine ligands are positioned in their binding pocket. We also discuss a newly postulated modulatory site for benzodiazepines at the α(1)/β(2) subunit interface, homologous to the classical benzodiazepine binding pocket.

Diazepam Decreases Action Potential Firing of Neocortical Neurons via Two Distinct Mechanisms

Benzodiazepines are widely used in clinical anesthesia as premedication, but also to induce general anesthesia. Recent in vitro studies suggest that γ-aminobutyric acid type A receptors, harboring a classical high-affinity benzodiazepine binding site, possess another "nonclassical" binding site for benzodiazepines. At present, it is unclear if, and to what extent, this novel nonclassical binding site is of relevance for the actions of benzodiazepines in the central nervous system. Because neocortex is involved in mediating the sedative and hypnotic properties of general anesthetics, we quantified the actions of diazepam over a wide range of concentrations (from 10 nM up to 100 μM) in organotypic slice cultures using extracellular multiunit recordings of spontaneous action potential activity. Up to a concentration of 6.25 μM, diazepam reduced the activity of neocortical neurons, approaching a maximum of approximately 20%. This action was nullified by the benzodiazepine antagonist flumazenil. At concentrations >12.5 μM, diazepam evoked a second concentration-dependent dampening of network activity. Unlike the low concentration effect, this high concentration component was resistant to flumazenil. Diazepam induced a biphasic attenuation of spontaneous action potential firing of neocortical neurons. Low to moderate concentrations caused a monotonic, mild depression that is mediated via the classical binding site as it is antagonized by flumazenil. However, the effects of diazepam observed at high concentrations were not affected by flumazenil. Hence, these findings support the concept of at least 2 different binding sites for benzodiazepines on γ-aminobutyric acid type A receptors. Furthermore, our results are consistent with the hypothesis that the classical high-affinity binding site mediates low-dose diazepam actions, such as amnesia, anxiolysis, and sedation, while a second, nonclassical and independent site contributes to the anesthetic effects of diazepam, such as hypnosis and immobility.

Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

GABAA receptor populations with novel subunit combinations and drug binding profiles identified in brain by alpha 5- and delta-subunit-specific immunopurification.

The pharmacological significance and structural basis of the gamma-aminobutyrate (GABAA) receptor heterogeneity was investigated in situ by an immunobiochemical analysis of receptor populations characterized by the delta- and alpha 5-subunit. Using an antiserum specific for the delta-subunit, a population of GABAA receptors (21 +/- 2% of solubilized receptors) was immunoprecipitated from rat brain extracts which contained high affinity benzodiazepine binding sites. They were distinguished from those immunoprecipitated by the alpha 1- and alpha 3-subunit antisera by a 4-5-fold and 5-10-fold higher affinity for diazepam and beta CCM, respectively. Using the delta-antiserum in immunoaffinity chromatography the delta-subunit was found to be associated with the alpha 1-, alpha 3-, beta 2/3-, and gamma 2-subunits, suggesting that the latter conveys benzodiazepine receptor sensitivity also to GABAA receptors containing the delta-subunit. The receptor population immunoprecipitated by the alpha 5-subunit antiserum (10 +/- 2% of receptors solubilized from whole brain extracts) was characterized by affinities for zolpidem, beta CCM, and CL 218872 which distinguished it from all other known receptor populations. The alpha 5-subunit was associated with the alpha 1-, alpha 3-, beta 2/3- and gamma 2-subunits pointing to differential subunit combinations. Indeed, when receptor populations were immunoprecipitated by the alpha 5-subunit antiserum from different brain regions, zolpidem displayed striking differences in affinity pointing to the role of subunits other than alpha 5 in determining receptor affinity.

The postnatal development of benzodiazepine receptor sites in the chick optic lobe is modulated by environmental lighting

The present paper describes the ability of benzodiazepin receptor sites to undergo light mediated-plastic changes during the early postnatal development of the chick optic lobe. The postnatal development pattern of these receptors was studied under different levels of light stimulation, i.e. normal-, light- and dark-rearing. At hatching the specific binding of [ 3H]Flunitrazepam was 0.23 ± 0.01 pmol/mg protein. The developmental profile shows a sharp and transient peak of receptor overexpression between the 1st and the 2nd postnatal day in the three experimental groups. Between the 2nd and the 6th day significant differences were found between the three groups, being this difference maximal during the peak of over-expression. In fact, on the 2nd day the specific [ 3H]Flunitrazepam binding showed an increase of 17% ( P < 0.0005) and a decrease of 34% ( P < 0.0005) for light- and dark-reared animals as compared with normally-reared ones. The changes in receptor density were transient since from the 6th day onward they gradually disappeared, being almost identical in the three groups by the day 15. At this moment the number of benzodiazepine receptor sites stabilized at the adult level. Scatchard analysis at the 2nd postnatal day revealed that the differences observed in the high affinity benzodiazepine binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.76 ± 0.03, 3.40 ± 0.01 and 1.46 ± 0.11 pmol/mg protein in normally-, light- and dark-reared chicks, respectively; while the apparent dissociation constants were unaffected. The present work shows that variations in the light intensity during the early postnatal life affect the postnatal developmental pattern of benzodiazepine receptor sites. This effect is maximal at the time of appearance of the peak of receptor overexpression (1–2 postnatal day) suggesting that the peak corresponds to a brief period of high plasticity for the receptor development.

Identification and immunohistochemical mapping of GABA A receptor subtypes containing the δ-subunit in rat brain

Synaptic inhibition in brain is mainly mediated via GABA A receptors which display a striking structural heterogeneity. A novel type of GABA A receptor subunit, the δ-subunit, has recently been described based on molecular cloning of its cDNA [1]. To identify the prevalence and distrubution of GABA A receptors which contain the δ-subunit protein in situ, polyclonal site-directed antisera were developed against three synthetic peptides derived form the rat δ-subunit cDNA-sequence. All antisera specifically recognized a 54 kDa protein in GABA A receptor preparation. Nearly 30% of the GABA A receptors contained the δ-subunit immunoreactivity and displayed high affinity GABA and high affinity benzodiazepine binding sites as shown by immunoprecipitation. Receptors which contain the δ-subunit were immunohistochemically shown to be restricted to a few brain areas such as the cerebellum, thalamus and dentate gyrus of the hippocampal formation. Thus, those neurons which express GABA A receptors with a δ-subunit have now been visualized and made accessible for a functional analysis of this GABA A receptors subtype in situ.

Antiproliferative Action of Benzodiazepines in Cultured Brain Cells Is Not Mediated Through the Peripheral‐Type Benzodiazepine Acceptor

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.

High-Affinity Benzodiazepine Binding Sites on Rat Peritoneal Mast Cells and RBL-1 Cells: Binding Characteristics and Effects on Granule Secretion

High-affinity binding sites for benzodiazepines are present in a wide range of tissues other than brain, and appear to be pharmacologically distinct from central nervous system receptors. Binding sites have been reported on rat peritoneal mast cells, but specificity of binding and functional effects have not been addressed in these cells. Using the specific radioligand [3H]-Ro5-4864, which binds to peripheral but not central binding sites, we characterized binding to rat peritoneal mast cells and the related cell line, RBL-1 cells, and effects on granule secretion. Studies in rat peritoneal mast cells disclosed one class of saturable binding sites with a dissociation constant of KD = 5.29 +/- 1.26 nM (mean +/- SE) and maximal binding Bmax = 357 +/- 113 fmol/10(6) cells. Competition studies showed that IC50 for Ro5-4864 was 15.7 nM, for PK11195 was 10.9 nM, for diazepam was 191 nM, and for clonazepam was greater than 10(-5) M. Studies in RBL-1 cells also showed one class of saturable binding sites with KD = 1.11 +/- 0.72 nM and Bmax = 177 +/- 69 fmol/10(6) cells; specificity was similar to peritoneal mast cells. Incubation of peritoneal mast cells with Ro5-4864 (10(-10)-10(-5) M) for 60 min, or with 10(-6) M for 0-60 min, showed no change in histamine or serotonin release either in unstimulated mast cells or those stimulated by compound 48/80 or substance P. Experiments with the fluorescent dye Quin-2 showed no change in free intracellular calcium levels in peritoneal mast cells after incubation with Ro5-4864 ranging from 10(-10) to 10(-6) M.

'High-affinity' peripheral-type benzodiazepine-binding sites.

1 ,4-Benzodiazepines are widely prescribed as minor tranquillizers or anxiolytics, as sedative-hypnotics and anticonvulsants. Some or possibly all of these effects may be explained by these compounds potentiating the action of the inhibitory neurotransmitter, y-aminobutyrate (GABA) (for reviews, see Turner & Whittle, 1983; Haefely et al., 1985). Thus some clinically active benzodiazepines potentiate neuronal postsynaptic GABA-receptor mediated chloride ion influx leading to membrane hyperpolarization. This receptor has been purified and shown to be an oligomeric complex of two protein subunits with binding sites for GABA and benzodiazepines (for reviews, see Turner & Whittle, 1983; Haefely et al., 1985). High-affinity benzodiazepine-binding sites were first reported by Braestrup & Squires (l977), who showed highaffinity diazepam-binding sites in brain and peripheral tissues. Central and peripheral binding sites could be distinguished from each other by (a) both types having high affinity for diazepam, (b) central sites having high affinity for clonazepam but low affinity for Ro-5-4864 (4’chlorodiazepam), (c) peripheral sites having low affinity for clonazepam and high affinity for Ro-5-4864. These peripheral-type sites were subsequently found in brain (Richards et al., 1982; Shoemaker et al., 1983) and have been proposed to be glial on the basis of neuronal cell lesioning studies (Schoemaker et a[., 1982). Several functions have been proposed for these peripheral/ non-neuronal benzodiazepine high-affinity binding sites, for example, in prostaglandin D, formation (Majewska & Chuang, 1985) in Ca2+ channel function (Doble et al., 1985) and in regulation of cell proliferation (Wang et al., 1984). We have previously reported (O’Beirne & Williams, 1984) that whereas we could measure high levels of peripheraltype benzodiazepine high-affinity binding to platelets from rodents, we were unable to detect this binding to platelets from non-rodents, and that this ‘lack’ of binding was probably not due to either states of activation nor presence of inhibitors or lack of activators. Whereas high-affinity binding sites of the peripheral-type could be shown using [3H]Ro-5-4864 or [3H]flunitrazepam, when membranes from rat kidney, liver and brain were, assayed, no specific binding of the peripheral-type could be shown to membranes from ox kidney, liver or brain nor human post-mortem membranes from kidney, liver or cerebral cortex. We conclude that in these tissues in these species peripheral-type binding is absent, immeasurably low or of low affinity. In rat brain, subcellular fractionation by differential centrifugation (Laduron et al., 1983) showed the nuclear fraction to have greatest enrichment of ‘peripheral-type’ binding sites. When brain fractions were resolved further using sucrose isopycnic density gradient centrifugation, for the ‘N’ fraction the binding sites were found enriched at an

The peripheral high affinity benzodiazepine binding site is not coupled to the cardiac Ca 2+ channel

Ro 5-4864, the prototype ligand of the peripheral benzodiazepine binding site caused a decrease of the action potential duration in isolated guinea-pig cardiac myocytes. Voltage-clamp experiments showed that, at concentrations below 3 X 10(-6) M, Ro 5-4864 caused a parallel outward shift of the membrane current elicited by depolarization to + 10 mV from a holding potential of -50 mV. The peak inward Ca2+ current (ICa) and the inwardly rectifying K+ current were not affected. ICa was reduced by Ro 5-4864 at concentrations above 3 X 10(-6) M. At these concentrations, Ro 5-4864 also caused a negative inotropic effect in isolated guinea-pig papillary muscles, reduced K+ depolarization-induced contractures of the isolated rat aorta and inhibited [3H]nitrendipine binding to guinea-pig cardiac membranes. These data provide no evidence that the peripheral high affinity benzodiazepine binding site is coupled to the cardiac Ca2+ channel. The possibility cannot be excluded that a postulated micromolar affinity benzodiazepine receptor is associated with the Ca2+ channel.

Reduction in benzodiazepine receptors associated with Purkinje cell degeneration in ‘nervous’ mutant mice

HIGH affinity benzodiazepine binding sites which are both stereospecific and saturable1–3 have recently been characterised in the mammalian central nervous system (CNS). The good correlations obtained between the binding affinities of a series of benzodiazepines and their potencies as anticonvulsants, anxiolytics and muscle relaxants strongly suggest that these sites may function as pharmacological receptors for the benzodiazepines1,4. Although both the gross anatomical3,5 and subcellular distribution6 of these sites have been described, the cellular localisation of these sites is not known, nor has a direct functional link been established between benzodiazepine binding sites and the neuropharmacological effects of these drugs. We now report that ‘nervous’ mutant mice, which undergo a selective loss of more than 90% of their cerebellar Purkinje cells during maturation7,8, have a marked reduction in the number of cerebellar benzodiazepine receptors. This loss in benzodiazepine receptors seems to be confined to the cerebellum, and is not accompanied by an alteration in the affinity of the receptor for diazepam. These data clearly demonstrate the loss of benzodiazepine receptors in a cell type which has been shown to be both neurochemically9 and electrophysiologically10 sensitive to benzodiazepines, thus providing evidence that benzodiazepine receptors function in the pharmacological actions of these drugs.