Abstract
Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.
Highlights
The fast neuronal action of the major inhibitory neurotransmitter, ␥-aminobutyric acid (GABA),2 is mediated by GABA type A (GABAA) receptors
The understanding of the relative positioning and overlap of modulators with different allosteric properties is essential for the rational design of ligands of the benzodiazepine site in different isoforms of the GABAA receptor
We present here data on the overlap of positive classical benzodiazepines with imidazobenzodiazepines in ␣12␥2 receptors
Summary
Synthesis—We used a substance in the imidazobenzodiazepine series we call the Imid-NCS compound, which is similar to Ro15-4513 except that the –N3 group is replaced with –NCS (see Fig. 1). To protect the binding site from covalent reaction, membranes were incubated with unlabeled Ro15-1788 or flunitrazepam for 10 min on ice. Subsequently, the Imid-NCS compound was added, and 3 min later, the first centrifugation was started. Expression and Functional Characterization in Xenopus Oocytes—Xenopus laevis oocytes were prepared, injected, and defollicated as described previously [28, 29] They were injected with 50 nl of cRNA solution containing wild-type or mutant ␣1, 2, and ␥2 subunits at a concentration of 10:10:50 nM [30] and incubated in modified Barth’s solution (10 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.34 mM Ca(NO3)2, 0.41 mM CaCl2, 100 units/ml penicillin, and 100 g/ml streptomycin) at ϩ18 °C for at least 24 h before the measurements. Mutant receptors were treated for 1 min with 1 M Imid-NCS compound
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