Peripheraltype Benzodiazepine Receptors Pbrs Pharmacological and Biochemical Characteristics

Benzodiazepines are widely used for their anxiolytic, anticonvulsant, and hypnotic actions. It has been well established that the major pharmacological effects of benzodiazepines are mediated by the y-aminobutyric acid (GABA)a receptors in the CNS (1,2). However, in search of specific binding sites for benzodiazepines outside the CNS another class of binding sites was first observed in the kidney (3) and later determined to be present in apparently all tissues including the CNS (4-7). This class of binding sites is commonly referred to as the peripheral-type benzodiazepine recognition sites or receptors (PBRs) owing to its initial discovery in peripheral tissues.

PBRs were distinguished from GABAA/benzodiazepine receptors by several criteria. Although both receptors bind diazepam with relatively high affinity, they exhibited very different binding specificities. In rodent species, PBRs bind 4'-chlorodiazepam with high affinity, whereas GABAa receptors show low affinity for this benzodiazepine (6,7). Conversely, clonazepam and flumazenil, which bind with high affinity to GABAa receptors, exhibit very low affinities for PBR. PBR also bind with high affinity the imidazopyridine alpidem and has a low affinity for the imidazopyridine zolpidem (8). On the contrary, GABAa receptors bind with high-affinity zolpidem and have low affinity for alpidem. In addition, PBRs have high affinity for three classes of compounds, isoquinoline (9), indoleacetamide (10), and pyrrolobenzoxazepine (11) derivatives, which do not bind to the GABAa receptors (10,11). Isoquinolines were the major tool used for the identification and characterization of PBR (5-7). In addition to these differences in drug specificity, it has been well-established that GABAA/benzodiazepine receptors, composed of50-55 kDa protein subunits, are coupled to synaptosomal chloride channels whereas PBR, an 18 kDa protein associated with other mitochondrial proteins, are not coupled to GABA recognition sites and their function will be addressed in this review. Subcellular fractionation studies demonstrated that PBRs were primarily localized on mitochondria (12-14), and more specifically on the outer mitochondrial membrane (15), although it is likely that they are not exclusive to this organelle. A plasma membrane location for this receptor was recently identified (16-18).

The first identification of a molecular component associated with PBRs was made possible by the development of a photoaffinity probe, the isoquinoline propanamide PK 14105 (19). This probe specifically labeled an 18 kDa protein, which was subsequently purified (20,21), and the corresponding cDNA cloned from rat (22), human (23,24), bovine (25), and murine (26) species. The cDNA sequence of the 18 kDa protein specifies an open reading frame of 169 aminoacids rich in tryptophan residues, with high-sequence homology (>80%) across species. Expression studies with the cDNA probes demonstrated that the 18 kDa protein contains the binding domains for PBR ligands although, owing to the constitutive expression of PBRs in all cells used for transfections, the presence of other (PBR-associated) proteins important for PBR ligand-binding expression cannot be excluded. In support of this hypothesis, we should consider that although high-affinity isoquinoline binding is diagnostic for PBRs, the affinity of benzodiazepines for PBRs is species-specific, varying from high affinity (rodents) to low affinity (bovine) (6,7). These species differences in benzodiazepine binding may be also owing to either structural differences in the 18 kDa protein or to differences in the components comprising the PBR complex in the mitochondrial membranes.

No other mammalian protein sharing homology with the 18 kDa protein was identified. However, a 32% amino-acid identity (66% when accounting for conserved substitutions) was found with the tryptophan-rich-sensory-protein tspO (also called crtK), involved in carotenoid biosynthesis in Rhodobacter capsulatus and Rhodobacter sphaeroidesphotosynthetic bacteria (27,28). The gene encoding the 18 kDa PBR protein has been isolated and characterized for rat (29) and human (24). In both species, the gene contains four exons spanning 10-13 kb and the locations of the introns are identical.

One of the interesting features of the 18 kDa PBR protein is the observation that, although this protein is targeted to the mitochondria, it does not contain a typical mitochondria-targeting signal sequence. The amino-terminal sequence of the 18 kDa PBR protein is hydrophobic and resembles a signal peptide, but it is not cleaved when the protein is incorporated in the mitochondrial membrane. In contrast, the carboxy-terminal sequence is hydrophilic, suggesting that it is exposed to the cytoplasmic environment.

We isolated and characterized the 18 kDa PBR cDNA from MA-10 Leydig cells (26). Expression of this cDNA in mammalian cells resulted in an increase in the density of both benzodiazepine and isoquinoline binding sites. In order to examine whether the increased drug binding is owing to the 18 kDa PBR protein alone or to other constitutively expressed components of the receptor, an in vitro system was developed using recombinant PBR protein (26). Isolated maltose-binding protein (MBP)-PBR recombinant fusion protein incorporated into liposomes—formed using lipids found in steroidogenic outer mitochondrial membranes, but not MBP alone—maintained its ability to bind isoquinolines, but not benzodiazepines. Addition of mitochondrial extracts in the liposomes resulted in the restoration of benzodiazepine binding. The protein responsible for this effect was then purified and identified as the 34 kDa voltage-dependent anion channel (VDAC) protein, which by itself does not express any drug binding. Interestingly, a number of laboratories have identified a 30-35 kDa protein, nonspecifically labeled using irreversible isoquinolines and benzodiazepines, to be associated with PBR (4-7). Among the ligands used to identify this 30-35 kDa protein was flunitrazepam (4-7). Based on the observation that the 35 kDa protein photolabeled with flunitrazepam could also bind radiolabeled dicyclohexylcarbodiimide, a reagent that covalently binds to VDAC, and that specific reagents which inhibit VDAC function were able to abolish PBR ligand binding, the hypothesis that VDAC was part of PBR was advanced (4). Moreover, we observed that, among the PBR ligands tested, only flunitrazepam could specifically antagonize the hormone-stimulated cholesterol transport and steroidogen-esis, acting via PBR (30). Furthermore, the observation that the 18 kDa PBR was isolated as a complex with the 34 kDa VDAC and the inner mitochondrial membrane adenine nucleotide carrier (ADC) (31) suggested that PBR is not a single protein receptor but a multimeric complex.

These studies demonstrated that VDAC is associated functionally to the 18 kDa PBR and is part of the benzodiazepine binding site in the PBR. Benzodiazepine binding, however, will be expressed only in the presence of the 18 kDa PBR protein that confers the other part of the recognition site. This model is also in agreement with the finding that the species difference in benzodiazepine binding may be owing to a five nonconserved amino acids in the C-terminal end of the 18 kDa PBR protein (32). Although the 18 kDa PBR and VDAC are required for drug binding, we cannot exclude the possibility that in vivo other proteins may be transiently or permanently associated with the PBR complex and modulate drug binding in an "allosteric" manner.

VDAC is a large-conductance large-diameter, about 3nm, ion channel with thin walls formed by a |3-sheet structure and located in the outer mitochondrial membrane, especially in the junctions between outer and inner membranes (contact sites) where it may complex with the adenine nucleotide carrier, hexokinase, and creatine kinase (33). VDAC forms a slightly anion-selective channel with complex voltage dependence and has been referred to as "mitochondrial porin" by analogy to bacterial porins. VDAC is believed to allow transport of metabolites and small molecules between the cytoplasm and the inner mitochondrial membrane (33,34).

Considering the interaction of 18 kDa PBR with VDAC at the contact site level, we will have to consider a potential role of other proteins shown to participate in contact site formation. The inner mitochondrial membrane ADC was previously shown to be structurally associated with PBR components (31). The inner mitochondrial megachannel (IMC) is also located in the inner membrane of the contact sites and represents activities regulated by voltage and ion (i.e., calcium) changes that result in pore opening and permeability increases (35,36). IMC is inhibited by PBR ligands (37) and is sensitive to the immunosuppressant cyclosporin A (35). Interestingly, we observed that cyclosporin A is a noncompetitive inhibitor of PBR, suggesting that IMC may regulate PBR in an "allosteric" manner (Papadopoulos, unpublished data). Taken together, these observations we propose a model of the PBR complex, present at the contact sites of mitochondrial membranes, composed of the 18 kDa PBR protein, VDAC, and two inner membrane proteins, ADC and IMC.

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