Affinity of various benzodiazepine site ligands in mice with a point mutation in the GABAA receptor γ2 subunit
Introduction
GABAA receptors are ligand gated ion channels that can be modulated by a large variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neurosteroids, anesthetics and convulsants [1]. A variety of evidence indicates that these compounds exert their action via distinct allosteric binding sites on these receptors. GABAA receptors are composed of five subunits forming the chloride ion channel that can be opened by GABA [2], [3]. So far, at least 19 different subunits belonging to different subunit classes (α, β, γ, δ, ɛ, π, θ, ρ) have been identified in the mammalian brain [4], [5]. The majority of GABAA receptors are composed of 2α, 2β, and 1γ subunit. Using the crystal structure of the acetylcholine binding protein [6] as a template for comparative modeling, homology models of the extracellular domain of GABAA receptors were recently constructed that for the first time provided insights in the topography of the GABA and benzodiazepine binding site of these receptors [7], [8]. Whereas the two GABA binding sites of these receptors are located at the β+α-interface, the benzodiazepine binding site is located at the α+γ-interface of GABAA receptors. Computational docking in models of the benzodiazepine site presently is hampered by model uncertainty leading to unclear side chain positions. In some cases, experimental evidence can possibly discriminate between different ligand positions. For instance, possible dockings of Ro 15-4513, an imidazobenzodiazepine used as photoaffinity label, were discussed recently [9], [10].
Various point mutations have been generated in recombinant receptors to identify amino acid residues important for interactions with benzodiazepine binding site ligands [3], [11]. The first residue identified to be of importance for the binding of benzodiazepines was the histidine in position 101 of the α1 subunit (α1H101). Mutation of this residue to arginine (α1H101R) dramatically reduced the affinity of classical benzodiazepines, such as diazepam or flunitrazepam, but did not impair the affinity of the imidazobenzodiazepines Ro 15-1788 or Ro 15-4513 [12], [13]. In contrast, the affinity of these imidazobenzodiazepines as well as that of flunitrazepam was strongly impaired by substituting tyrosine at the position 209 of the α1 subunit by glutamine (α1Y209Q) [13]. In addition, using [3H]flunitrazepam or [3H]Ro 15-4513 as a photolabel, residues α1H101 [14], [15] or α1Y209 [9] were irreversibly labeled, respectively, supporting the conclusion that these residues are located in the benzodiazepine binding site of GABAA receptors and can be contacted by benzodiazepine site ligands with different structure.
In line with the conclusion that the benzodiazepine binding site is formed at the interface of α and γ subunits, several mutations in the γ2 subunit of GABAA receptors also led to changes in the affinity of various benzodiazepine site ligands [11]. Thus, for instance, the γ2F77I mutation dramatically reduced the affinity of zolpidem, Ro 15-1788, DMCM and Cl 218872 for recombinant α1βγ2F77I receptors, but only weakly reduced the affinity of classical benzodiazepines, such as diazepam or flunitrazepam [16], [17]. Interestingly, homology models of GABAA receptors indicate that residues α1H101 from the so-called loop A, α1Y209 from the loop C, and residue γ2F77 from loop D are situated approximately equidistantly spaced around a solvent-accessible pocket and do indeed line this pocket (Fig. 1, [8]).
Performing binding studies with recombinant receptors, especially when using transiently transfected HEK cells, is rather cumbersome due to the large amounts of cell culture plates needed for dose-response curves. To further investigate the benzodiazepine binding pocket and its interaction with compounds from different structural classes, we here took advantage of the recently generated mice that carry the γ2F77I point mutation [18]. Using brain membranes from γ2F77I or C57BL/6J mice we investigated the potency of 24 benzodiazepine binding site ligands from 16 different structural classes for inhibition of [3H]flunitrazepam binding. Results obtained identify compounds from two additional structural classes that are strongly influenced by this mutation.
Section snippets
Chemicals
[3H]flunitrazepam (84.5 Ci/mmol) was purchased from Perkin-Elmer Life Sciences. Compounds (see Table 1 and Fig. 2, Fig. 3 for structures) were obtained from the following sources: flunitrazepam (7-nitro-1,3-dihydro-1-methyl-5-o-fluorophenyl-2H-1,4-benzodiazepin-2-one), diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one), flurazepam (7-chloro-1,3-dihydro-1-ethylaminodiethyl-5-o-fluorophenyl-2H-1,4-benzodiazepin-2-one), midazolam
Results
Brain membranes from γ2F77I mice and littermate controls were incubated with various concentrations of [3H]flunitrazepam in the absence or presence of 10 μM diazepam and binding data obtained were subjected to Scatchard analysis. Kd values for [3H]flunitrazepam binding were 7.9 ± 0.2 and 3.8 ± 0.4 for γ2F77I mice and littermate controls, respectively, indicating a 2-fold shift in the affinity of flunitrazepam in γ2F77I mice. Interestingly, the Bmax values were 4373 ± 290 and 3330 ± 198 (mean ±
Discussion
In the present study the potency for inhibition of [3H]flunitrazepam binding of 24 benzodiazepine binding site ligands from 16 different structural classes was investigated in mice carrying the point mutation γ2F77I. These mice have been demonstrated previously to behave normally in a range of standard behavioural tests and the overall architecture of the brains also appeared normal [18] as expected from a point mutation that only weakly reduced GABA affinity of the receptor [20]. In contrast
Acknowledgements
This work was supported by projects P14385-Gen and P16397 of the Austrian Science Fund to W.S. and VolkswagenStiftung grant I/78554 to W.W.
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