Identifying agonistic and antagonistic mechanisms operative at the GABA receptor

Abstract

Based on our molecular modeling investigations of the glycinergic receptor, we expanded our studies to similarly investigate the GABAergic receptor. New data suggest there may exist a slightly different agonistic mechanism for the molecules described herein as compared to glycine. The origin of this is undoubtedly the fact that, while glycine has a positive and two negative binding sites, it is significantly shorter than GABA and the other GABA agonists. Clearly, discovery of more glycine agonists is needed to further clarify this point. Moreover, we find a remarkedly different antagonistic mechanism exists for this phylogenetically newer inhibitory system in the central nervous system (CNS) than recently reported for strychnine and eight weaker glycine antagonists. We used GABA and six agonists (muscimol, dihydromuscimol, THIP, isoguvacine, trans-3-aminocyclopentane-1-carboxylic acid, piperidine-4-sulfonic acid) and five antagonists (bicuculline-N15-methobromide, R5135, pitrazepin, iso-THAZ and securinine) to derive our conclusions. We found that each of the agonists have three clearly defined atoms that can serve as attachment points at the GABAA receptor site. One of the three attachment atoms includes a carbonyl or carboxylate oxygen. The role of the carbonyl or carboxylate atom is very important. First, we theorize that a rapid two-point attachment occurs (one from the positive end and one from one of the other two negative atoms on the ligand) at the recognition site in the receptor where GABA or a GABAergic agonist binds. The positive end of the agonist perhaps associates through hydrogen bonding to a beta-carboxyl group in one of the aspartate molecules in the polypeptide. The negative attachment points perhaps bind through hydrogen bonding to arginine molecules in this polypeptide. The second negative site in the agonist immediately triggers a conformational change by pulling together the aforementioned groups by electrostatic attraction, and hence opening the chloride channel. We propose the carbonyl oxygen is partly responsible for triggering the opening by formation of a double hydrogen bond to arginine. We postulate that this attraction is the first step inducing the conformational change. In the case of the GABA antagonists investigated, a fourth attachment site was not found. In fact only two sites have been identified similar to the group II glycine antagonists. Our data support a hypothesis for GABAergic antagonist activity which suggests that the antagonist simply binds to the recognition site and blocks the neurotransmitter, GABA, from entering this site thereby preventing the opening of the chloride channel; it just stays closed. This mechanism is different from the mechanism proposed for the large number of Group I glycine antagonists (Aprison et al.: J Neurosci Res 41: 259-269, 1995).

Receptor-Independent Activation of GABAergic Neurotransmission and Receptor-Dependent Nontranscriptional Activation of Phosphatidylinositol 3-kinase/Protein Kinase Akt Pathway in Short-Term Cardiovascular Actions of Dexamethasone at the Nucleus Tractus Solitarii of the Rat

“…The selectivity of bicuculline methiodine and 2-OH saclofen as GABA A and GABA B receptor antagonists (AlDahan et al, 1990;Galvez-Ruano et al, 1995), or AMD as an RNA synthesis inhibitor , has been documented. Activation of type II, but not type I, glucocorticoid receptors contributes to hypertension induced by the agonists (Clapham and Turner, 1997).…”

“…These coordinates were selected to cover subdivisions of NTS in which functionally identified barosensitive neurons reside (Chan and Sawchenko, 1998). Test agents used included a synthetic glucocorticoid, Dex (Sigma-Aldrich, St. Louis, MO); a selective glucocorticoid type II receptor antagonist (Clapham and Turner, 1997;Limbourg et al, 2002), mifepristone (RU486; SigmaAldrich), that does not bind to the type I mineralocorticoid receptor or to aldosterone receptors (Brogden et al, 1993); a selective GABA A receptor antagonist (Galvez-Ruano et al, 1995), bicuculline methiodine (Sigma-Aldrich); a GABA B receptor antagonist (Al-Dahan et al, 1990), 2-hydroxy saclofen (2-OH saclofen; Sigma-Aldrich); a nonselective nitric-oxide synthase (NOS) inhibitor (Reif and McCreedy, 1995), N G -monomethyl-L-arginine acetate (L-NMMA; SigmaAldrich); a PI3K inhibitor (Vlahos et al, 1994), LY294002 (Calbiochem, San Diego, CA); and an RNA synthesis inhibitor , actinomycin D (AMD; Calbiochem). The dose and treatment scheme were adopted from previous reports (Clapham and Turner, 1997;Ouyang and Wang, 2000;Hafezi-Moghadam et al, 2002;Limbourg et al, 2002;Moreno et al, 2002;Chan et al, 2003), which used the same test agents for the same purpose as in this study.…”

Securinega Alkaloids from Flueggea leucopyra

“…5 showed an [MϩH] ϩ ion peak at m/z: 280.1540 (Calcd for C 15 C-NMR data of 5 (Table 1) were very similar to those of secu'amamine B (4), 8) suggesting that 5 was a dihydrosecurinine type alkaloid with (Fig. 3) indicated that the relative configuration at C-2 of 5 was opposite to those of 3 and 4.…”

“…13) Pharmacology investigations indicated that securinine was a stereospecific GABA A receptor antagonist with a significant central nervous system (CNS) activity. 14,15) In searching for bioactive alkaloids from the Euphorbiaceae plants, we had isolated some chemical constituents from Securinega suffruticosa and Flueggea virosa. 3,4) Flueggea leucopyra was a shrub which only distributed in Sichuan and Yunnan Provinces of China.…”

A mutant residue in the third transmembrane region of the GABAA alpha1 subunit causes increased agonistic neurosteroid responses

“…Piperidine-4-sulfonic acid (P4S), a specific GABA A partial agonist used in basic electrophysiology experiments (Krossgaard-Larsen et al ., 1980; Galvez-Ruano et al ., 1995) was dissolved in CFFR as a 100 mM stock and stored at 4°C. Before the experiment, P4S was diluted further in CFFR to the necessary concentrations.…”