Rat and Human Hippocampal a5 Subunit-Containing g- Aminobutyric AcidA Receptors Have a5b3g2 Pharmacological Characteristics

The g-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human a5b3g2 and a5b3g3 GABAA receptors stably expressed in L(tk) cells were characterized with the a5-selective ligand [H]L-655,708 and compared with the pharmacological characteristics of [H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [H]L-655,708 for a5b3g2 receptors (Kd 5 1.7 6 0.4 nM), compared with a5b3g3 receptors (Kd 5 15 6 3 nM). Rat and human hippocampal [ H]L655,708 binding sites had affinities of 2.2 6 0.6 and 1.0 6 0.2 nM, respectively, comparable to the affinity of a5b3g2 receptors. Pharmacological analysis of [H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for a5b3g2 but not a5b3g3 receptors. Immunoprecipitation of [H]L-655,708 binding sites from rat hippocampus with a g2selective antibody yielded 19 6 4% of total benzodiazepine binding sites measured using [H]Ro15–1788, whereas no specific binding was measured after immunoprecipitation with an anti-g3 antibody. Combinatorial immunoprecipitations of [H]muscimol binding sites with anti-a5 and anti-g2 or anti-a5 and anti-g3 antibodies established the preferential expression of a5g2 receptors, accounting for 22 6 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of a5b3g2 GABAA receptors in rat hippocampus, despite the clustering of a5 and g3 loci on the same chromosome. The GABAA receptor is the main inhibitory ligand-gated ion channel in the central nervous system. It contains modulatory sites for endogenous molecules such as the neurosteroids, as well as for many therapeutic drugs, such as barbiturates, anesthetics, and benzodiazepines (Sieghart, 1995). It is now generally accepted that the GABAA receptor is a pentameric protein with an integral chloride ion channel formed by the second transmembrane domain of each of the five subunits. A family of GABAA receptor subunits (a1–a6, b1–b3, g1–g3, d, and e) have been identified in mammalian brain using molecular cloning techniques (for review, see McKernan and Whiting, 1996; Davies et al., 1997; Whiting et al., 1997). At least one a subunit, one b subunit, and one g subunit are required to form fully functional receptors in vivo (Pritchett et al., 1989), and the combination of a and g subunits is a crucial determinant of the properties of the benzodiazepine binding site (Hadingham et al., 1993; Wafford et al., 1993; Luddens et al., 1994; Benke et al., 1996). The a1 subunit-containing receptors exhibit BZ1-type pharmacological characteristics, characterized by a high affinity for zolpidem, whereas a2, a3, and a5 subunits are present in BZ2type receptors, which have a low affinity for zolpidem (Pritchett et al., 1989; Pritchett and Seeburg, 1990). A third class of GABAA receptors also exists; these receptors contain an a4 or a6 subunit and have a low affinity for most of the classical benzodiazepines (Luddens et al., 1990; Wisden et al., 1991; Wafford et al., 1996; Benke et al., 1997). Receptors expressing an a5 subunit together with b and g2 subunits in cell lines are distinguished from BZ1 receptors by their low affinity for zolpidem (Pritchett and Seeburg, 1990; Luddens et al., 1994) and from other BZ2 receptors by their 10–20-fold higher affinities for Ro15–4513 (Hadingham et al., 1993; Luddens et al., 1994) and for several 8-substituted benzodiazepines (Gillard et al., 1994). Thus, a5-containing receptors have a unique pharmacological profile. In the rat central nervous system, a5 subunit-containing receptors have restricted and well defined expression. In situ hybridization and immunocytochemistry studies have shown that this subtype is present in abundance in the CA1 and CA3 fields of the hippocampus, is present to a lesser extent in ABBREVIATION: GABA, g-aminobutyric acid. 0026-895X/98/050928-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:928–933 (1998). 928 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from the cortex and olfactory bulb, and is virtually absent in other regions of the brain (Wisden et al., 1992; Fritschy and Mohler, 1995; Quirk et al., 1996). The exact subunit composition of receptors containing an a5 subunit in vivo is not known. The colocalization of a5, b3, and g3 subunits on chromosome 15 suggests possible associations among these subunits, and deletion of this locus reduces zolpidem-insensitive, radiolabeled benzodiazepine binding (Nakatsu et al., 1993). On the other hand, the pharmacological characteristics of a5-containing receptors immunoprecipitated from rat brain are closer to those demonstrated in cells transfected with a5b3g2 than those observed with a5b3g3 (McKernan et al., 1991; Luddens et al., 1994). Similarly, electrophysiological studies indicated a5b3g2L as the isoform expressed by hippocampal CA1 pyramidal cells (Burgard et al., 1996). Using a 50–100-fold selective ligand for a5 subunit-containing receptors (Quirk et al., 1996), [H]L-655,708, we describe here the pharmacological characteristics of a5 subunit-containing receptors in rat and human brain and compare them with those of stable cell lines expressing a5b3g2 and a5b3g3 receptors. Implications for the structure of native a5 subunit-containing receptors are discussed. Experimental Procedures Materials. [H]Ro15–1788 and [H]muscimol were purchased from New England Nuclear-Du Pont (Hertfordshire, UK). [H]L655,708 (76.7 Ci/mmol) was prepared as previously described (Quirk et al., 1996). CL218872 and CGS8216 were gifts from Lederle and Ciba-Geigy/Novartis, respectively, and other benzodiazepine site ligands were from Sigma Biochemicals or Research Biologicals Inc. Human tissues. Hippocampi were from adult cadaveric brain tissue obtained from subjects without any neurodegenerative disease and without obvious morphological abnormalities of the hippocampi. Membrane preparation and binding assays. P2 membranes were prepared from brain regions of adult male rats or from cadaveric hippocampi from adult human subjects as previously described (McKernan et al., 1991). Radioligand binding assays were performed with brain membranes or membranes prepared from stably transfected cell lines (Hadingham et al., 1993; Sur et al., 1997), with [H]L-655,708 (0.1–40 nM), in a final volume of 0.5 ml containing 50–100 mg of protein in 10 mM TriszHCl, 1 mM EDTA, pH 7.4, at 4°. For saturation analyses with rat and human hippocampi, 1 mM zolpidem was added to the assays, to prevent binding to other a subunits at high [H]L-655,708 concentrations. Similarly, immunoprecipitated receptor-protein A complexes were incubated with [H]L-655,708 (20–24 nM), [H]Ro15–1788 (20 nM), or [H]muscimol (40 nM) for 1–2 hr at 4°. Nonspecific binding was defined with 10 mM flunitrazepam or 100 mM GABA, for H-benzodiazepine or [H]muscimol binding, respectively. After 1–2-hr incubations at 4°, assay mixtures were filtered through Whatman GF/B filters using a cell harvester (Brandel) and were washed four times with cold buffer. Filters were immersed overnight in scintillation cocktail, and radioactivity was determined in a Beckman liquid scintillation counter. Data points were fitted by nonlinear regression analysis (Excel; Microsoft); for competition experiments, the Ki values were calculated according to the Cheng-Prusoff equation (Cheng and Prusoff,