Key Amino Acids in the γ Subunit of the γ-Aminobutyric Acid_AReceptor that Determine Ligand Binding and Modulation at the Benzodiazepine Site

Abstract

SUMMARYPharmacological analyses of ␥-aminobutyric acid A (GABA A ) receptor subtypes have suggested that both the ␣ and ␥ subunits, but not the ␤ subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different ␥ subunits in the receptor macromolecule to identify amino acids ␥2Phe77 and ␥2Met130 as key determinants of the benzodiazepine binding site. ␥2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-␤-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, ␥2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218,872, or methyl-␤-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of ␥1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that ␥2Phe77 and ␥2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the ␣ subunit, we demonstrated a critical role for the ␥ subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, ␥2Phe77 is homologous to ␣1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABA A receptor.The GABA A receptor, a member of the ligand-gated ion channel family, mediates synaptic inhibition through the gating of chloride ions, resulting in hyperpolarization of the cell membrane. It is the site of action of a number of pharmacological agents, including BZs, barbiturates, and anesthetics. The hetero-oligomeric receptor is formed from the coassembly of five different subunit classes [␣, ␤, ␥, ␦ (1, 2), and ⑀ (3, 4)] in a presumed pentameric arrangement (5, 6) to yield a family of receptor subtypes. It is the heterogeneity within these subunits that provides the molecular basis for the differences in pharmacology of receptor subtypes (7).Classic BZ pharmacology is exhibited by receptors containing a ␥2 subunit in combination with an ␣ and a ␤ subunit (8). The affinity of BZ ligands for the receptor is dependent on the ␣ subunit isoform, and hence compounds such as CL218,872 and zolpidem have higher affinity for ␣1␤n␥2 (n ϭ 1, 2, or 3) receptors than for other ␣ subunit-containing receptors (9, 10), and flunitrazepam and diazepam (11, 12) have very low affinity (Ͼ10 M) for ␣4␤n␥2 and ␣6␤n␥2. Mutagenesis studies have identified two amino acids on the ␣ subunit as contributing to the BZ binding site (13,14 McKernan et al. (16) that the ␥ subunit also contributes significantly to the BZ binding site. I...

Variations in Maternal Care Alter GABAA Receptor Subunit Expression in Brain Regions Associated with Fear

“…The inclusion of a g1 or g2 subunit in the GABA A receptor complex appears to define the presence of a BZ receptor site. Point mutations have been identified within both subunits that are sufficient to eliminate BZ receptor binding (Amin et al, 1997;Wingrove et al, 1997) and animals bearing a null mutation of the g2 subunit show approximately an 85% loss of [ (Gunther et al, 1995). Thus, the effect of maternal care on the expression of the g subunits might well contribute to the previously observed difference in BZ receptor-binding capacity between high and low LG-ABN mothers (Caldji et al, 1998).…”

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

“…The lack of complete disappearance of the tonic current in the hippocampus of ␦ Ϫ/Ϫ mice favors the involvement of other receptor subunits (15,62). Possible candidates are the ␥ 2 subunits since they are required for the observed facilitatory effect of FZP on tonic inhibition (42,43,(63)(64)(65)(66). However, it must be pointed out that benzodiazepine sensitivity can also be due to ␣ 5 subunit-containing receptors (67)(68)(69), which are largely expressed in the hippocampus (68 -71).…”

“…Tonic Currents from Untreated and Nocodazole-treated Neurons Are Sensitive to Flurazepam-To compare the pharmacology of tonic currents in untreated and nocodazole-treated neurons and to infer the subunit composition of extrasynaptic GABA A receptors, we studied the effect of benzodiazepines, known to be effective on ␥ 2 subunit-containing receptors (42,43). The efficiency of FZP was assessed by analyzing its effect on the peak amplitude and kinetics of sIPSCs.…”

Altered kinetics and benzodiazepine sensitivity of a GABA A receptor subunit mutation [γ 2 (R43Q)] found in human epilepsy

“…Interestingly, with this higher GABA concentration, we now found that diazepam and flunitrazepam potentiated currents in oocytes expressing the ␥ 2 (R43Q) subunit although to a significantly lesser extent than in those expressing WT ␥ 2 -subunit. Previous reports investigating amino acids involved in benzodiazepine binding and allosteric modulation have found differences in the residues and receptor subunits involved in binding benzodiazepines and imidazopyridines such as zolpidem (26,27), suggesting they act via different binding and perhaps transduction mechanisms. Therefore, we examined the ability of zolpidem to modulate ␥ 2 (R43Q)-containing GABA receptors.…”