[3H]L-655,708, a Novel Ligand Selective for the Benzodiazepine Site of GABAA Receptors which Contain the α5 Subunit

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Abstract

A compound (L-655,708) has been identified which has at least 50-fold selectivity for the benzodiazepine site on GABAA receptors containing an α5 subunit over those containing an α1, α2, α3 or α6 subunit in combination with β3 and γ2. The compound was radiolabelled with tritium and investigated as a novel radioligand which recognizes the benzodiazepine site of GABAA receptors which contain the α5 subunit. [3H]L-655,708 labels one saturable and specific population of binding sites in rat hippocampus with a Kd of 2.4 ± 0.7 nM and a Bmax of 256 ± 42 fmol/mg protein. The pharmacology of the binding site labelled was consistent with that of receptors present in cells transfected with α5, β2 and γ2 and with receptors immunoprecipitated from rat brain with an α5-selective antiserum. It is concluded that [3H]L-655,708 is the first radioligand to date which is selective for any BZ2 subtype of the GABAA receptor and should provide a valuable tool for elucidating the structure and function of the α5-containing GABAA receptor subtype. Copyright © 1996 Elsevier Science Ltd

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Materials

[N-methyl-3H]Ro 15-1788 (75.3 Ci/mmol) and [7,9-3H]Ro 15-4513 (27.4 Ci/mmol) were from DuPont/New England Nuclear. Flunitrazepam was from Sigma; Ro 15-4513, Ro 15-1788 and Zolpidem were from Research Biologicals, Inc.

Synthesis of L-655,708 and [3H]L-655,708

L-655,708 (Table 1) was prepared from 5-methoxyanthranilic acid by following the previously described procedure of Watjen et al. (1990). For the preparation of [3H]L-655,708, starting from 5-benzyloxyanthranilic acid, the same procedure was used to prepare the benzyl derivative (

Selectivity of L-655,708

Competition of L-655,708 for benzodiazepine sites labelled by [3H]Ro 15-1788 or [3H]Ro 15-4513 in stably transfected cells revealed it to be selective for cell lines containing the α5 subunit, as shown in Table 2. L-655,708 was found to be 107-fold, 61-fold, 54-fold and 184-fold selective for the α5-containing receptors over receptors containing α1, α2, α3 and α6 subunits, respectively. Consequently, the compound was tritiated to investigate whether it could be used as a radioligand.

Binding of [3H]L-655,708 to rat hippocampal membranes

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DISCUSSION

This study demonstrates that L-655,708 binds to α5-containing receptors with more than 50-fold higher affinity than to any other GABAA-receptors. This is more selective than analogues of Ro 15-4513, which previously have been shown to have some selectivity (Hadingham et al., 1993; Gillard et al., 1994) and, as such, is the most α5-selective compound reported to date. [3H]L-655,708 is shown to bind rapidly and selectively to α5-containing receptors with high affinity, and so is the first

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    We see two possible reasons for this underestimation of the tonic current component mediated by these receptors in our experiments. First, we have used a concentration of L-655,708 (20 nM) which is not fully saturating because at higher doses (e.g. >50–100 nM) the selectivity of this compound for this receptor type becomes questionable (Quirk et al., 1996; Atack et al., 2006; Vargas-Caballero et al., 2010). Second, it cannot be excluded that the effective concentration of this compound within a slice, around neurons from which recordings were made, was smaller than 20 nM due to limited perfusion through the slice tissue.

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