The M r 18,000 Subunit of the Peripheral-type Benzodiazepine Receptor Exhibits Both Benzodiazepine and Isoquinoline Carboxamide Binding Sites in the Absence of the Voltage-dependent Anion Channel or of the Adenine Nucleotide Carrier

Abstract

The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195. This receptor includes an M r 18,000 isoquinoline-binding subunit predominantly located in mitochondrial membranes. This protein has been found to copurify with two other mitochondrial proteins, namely the outer membrane voltage-dependent anion channel (VDAC), also known as mitochondrial porin, and the inner membrane adenine nucleotide carrier. In vitro reconstitution experiments suggested that the PBR was a multimeric complex in which the isoquinoline binding site was on the M r 18,000 subunit, denoted pk18, whereas the benzodiazepine binding site required the association of this subunit with VDAC to be expressed. Untransformed cells of the yeast Saccharomyces cerevisiae are devoid of specific binding sites for isoquinolines and benzodiazepines, whereas yeast cells transformed with a pk18-expressing vector exhibit RO5-4864 and PK11195 binding sites that are pharmacologically identical to those of the PBR. To clarify the role of VDAC and of the adenine nucleotide carrier, if any, in the constitution of the benzodiazepine binding site, yeast host strains were constructed in which the corresponding genes had been knocked out. Mitochondria prepared from pk18-producing cells devoid of either VDAC or adenine nucleotide carrier exhibit both benzodiazepine and isoquinoline carboxamide binding sites with little or no change in the K d values as compared with the wild-type background. These results rule out the contention that VDAC is indispensable for establishing the benzodiazepine binding site and are in agreement with the hypothesis that the M r 18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains.

Novel Androstenetriol Interacts with the Mitochondrial Translocator Protein and Controls Steroidogenesis

“…Hydropathy profile and experimental analysis have suggested that TSPO possesses a five-transmembrane structure (32,33), findings recently supported by electron cryomicroscopy of a bacterial homolog (34). Deletion of the C terminus of recombinant mammalian TSPO, which contains the CRAC motif, severely reduced cholesterol uptake when expressed in Escherichia coli, although PK 11195 binding was retained (25).…”

Mitochondrial Ion Channels

“…The binding of isoquinoline carboxamides and benzodiazepines is mutually competitive for the mBzR, and it was originally proposed that the binding site for benzodiazepines, such as the prototypical Ro 5-4864 (4′ Cl-diazepam), included the IBP, VDAC, and ANT (61). However, studies of yeast transformed with the IBP, but lacking either the VDAC or ANT protein, show high-affinity binding sites for isoquinoline carboxamides and benzodiazepines that are no different from binding sites of native receptors (62). More recently other proteins have been reported to associate with the IBP; these include the PBR-associated protein 1 [PRAX-1 (63)], the PBR-associated protein 7 [PAP7, or PKA with regulatory subunit 1α (64)], and a 10-kDa protein [pk10 (65)].…”

Two Binding Sites for [3H]PBR28 in Human Brain: Implications for TSPO PET Imaging of Neuroinflammation

“…The TSPO exists both as a monomer and as part of a multimeric complex involving several TSPO monomers with associated proteins, including the voltage-dependent anion channel (Boujrad et al, 1994(Boujrad et al, , 1996Rao and Butterworth, 1997). Although subunit interactions are not necessary for drug ligands to bind the TSPO monomer (Joseph-Liauzun et al, 1997;Lacapere et al, 2001), binding of some drugs to the TSPO is influenced by these interactions (Golani et al, 2001).…”