Flunitrazepam photoaffinity labeling of the GABAA receptor reduces inhibition of [3H]Ro15-4513 binding by GABA (original) (raw)
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Journal of Neurochemistry, 1992
In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.
Journal of Neurochemistry, 1992
In the present communication we have investigated the allosteric coupling between the y-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type 11) have been classically defined using CL 2 18.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high-and low-affinity binding sites for zolpidem was similar to that of type I and type I1 subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GA-BAA receptor and the BZD receptor subtypes was different. The high-and low-affinity binding sites for zolpidem seemed
Journal of …, 2001
Casula MA , Bromidge FA , Pillai GV , Wingrove PB , Martin K , Maubach K , Seabrook GR , Whiting PJ , Hadingham KL Neuroscience Research Centre, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, UK. Journal of Neurochemistry [2001, 77(2):445-451] Type: Journal Article, Comparative Study DOI: 10.1046/j.1471-4159.2001.00289.x The Digital Object Identifier (DOI) System enables identification of digital entities Abstract L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.
Neurochemistry International, 2008
Several studies have indicated a functional differentiation across the septotemporal axis of rat hippocampus. Our previous results have shown that the a 1 b 2 g 2 -GABA A receptor subtype dominates in dorsal hippocampus (DH), while the a 2 b 1 g 2 -subtype prevails in ventral hippocampus (VH). We therefore studied possible differences in the pharmacological properties and receptor binding parameters of the GABA A receptor subtypes between DH and VH, by examining: (1)(a) the specific binding of [ 3 H]-flunitrazepam (Benzodiazepine sites agonist) by using quantitative autoradiography, (b) the kinetic parameters of [ 3 H]-flunitrazepam specific binding, by using the ''wipe off'' technique and (2) the competitive displacement of [ 3 H]-flunitrazepam binding by using zolpidem (selective agonist of the a 1 -subtype) and L-655,708 (selective inverse agonist of the a 5 -subtype) and the enhancement of [ 3 H]-flunitrazepam binding by using etomidate (selective positive modulator of the b 2 -subunit), in an autoradiographical saturation kinetic study. Our results showed in VH compared to DH: (A) lower level of [ 3 H]-flunitrazepam binding, apparently due to weaker binding affinity (higher K D value), since no differences in the B max value could be detected, (B) higher IC 50 values for zolpidem and lower IC 50 values for L-655,708 and (C) higher EC 50 values for etomidate. In conclusion, the lower binding for zolpidem and etomidate and the higher binding for L-655,708 observed in VH support the evidence that the a 1 b 2 g 2 -GABA A receptor subtype dominates in DH and the a 5 -subtype prevails in VH. Further, our results suggest differential pharmacological effects of the benzodiazepines in DH compared to VH, with the sedative effects being more potent in the dorsal hippocampus. #
Selective labelling of diazepam-insensitive GABA A receptors in vivo using [ 3 H]Ro 15-4513
British Journal of Pharmacology, 2005
Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.