Activation of the Rat α1ß2ε GABAA Receptor by Orthosteric and Allosteric Agonists.

GABAA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABAA subunits cloned to date. GABAA receptors containing the ε subunit exhibit a significant degree of constitutive activity and have been suggested to be unresponsive to allosteric agents. In this study, we have characterized the functional properties of the rat α1ß2ε GABAA receptor. We confirm that the α1ß2ε receptor exhibits a higher level of constitutive activity than typical of GABAA receptors and show that it is inefficaciously activated by the transmitter and the allosteric agonists propofol, pentobarbital, and allopregnanolone. Manipulations intended to alter ε subunit expression and receptor stoichiometry were largely without effect on receptor properties including sensitivity to GABA and allosteric agonists. Surprisingly, amino acid substitutions at the conserved 9' and 6' positions in the second transmembrane (TM2) domain in the ε subunit did not elicit the expected functional effects of increased constitutive activity and resistance to the channel blocker picrotoxin, respectively. We tested the accessibility of TM2 residues mutated to cysteine using the cysteine-modifying reagent 4-(hydroxymercuri)benzoic acid and found a unique pattern of water-accessible residues in the ε subunit.

Analysis of Modulation of the ρ1 GABAA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5ß-reduced steroids, sulfated or carboxylated steroids, and ß-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5ßP), 3α5ßP sulfate, and ß-estradiol. The site for 3α5ßP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

Steady-state activation and modulation of the synaptic-type α1ß2γ2L GABAA receptor by combinations of physiological and clinical ligands.

The synaptic α1ß2γ2 GABAA receptor is activated phasically by presynaptically released GABA. The receptor is considered to be inactive between synaptic events when exposed to ambient GABA because of its low resting affinity to the transmitter. We tested the hypothesis that a combination of physiological and/or clinical positive allosteric modulators of the GABAA receptor with ambient GABA generates measurable steady-state activity. Recombinant α1ß2γ2L GABAA receptors were expressed in Xenopus oocytes and activated by combinations of low concentrations of orthosteric (GABA, taurine) and allosteric (the steroid allopregnanolone, the anesthetic propofol) agonists, in the absence and presence of the inhibitory steroid pregnenolone sulfate. Steady-state activity was analyzed using the three-state cyclic Resting-Active-Desensitized model. We estimate that the steady-state open probability of the synaptic α1ß2γ2L GABAA receptor in the presence of ambient GABA (1 µmol/L), taurine (10 µmol/L), and physiological levels of allopregnanolone (0.01 µmol/L) and pregnenolone sulfate (0.1 µmol/L) is 0.008. Coapplication of a clinical concentration of propofol (1 µmol/L) increases the steady-state open probability to 0.03. Comparison of total charge transfer for phasic and tonic activity indicates that steady-state activity can contribute strongly (~20 to >99%) to integrated activity from the synaptic GABAA receptor.

Steady-State Activation and Modulation of the Concatemeric α1ß2γ2L GABAA Receptor.

The two-state coagonist model has been successfully used to analyze and predict peak current responses of the γ-aminobutyric acid type A (GABAA) receptor. The goal of the present study was to provide a model-based description of GABAA receptor activity under steady-state conditions after desensitization has occurred. We describe the derivation and properties of the cyclic three-state resting-active-desensitized (RAD) model. The relationship of the model to receptor behavior was tested using concatemeric α1ß2γ2 GABAA receptors expressed in Xenopus oocytes. The receptors were activated by the orthosteric agonists GABA or ß-alanine, the allosteric agonist propofol, or combinations of GABA, propofol, pentobarbital, and the steroid allopregnanolone, and the observed steady-state responses were compared with those predicted by the model. A modified RAD model was employed to analyze and describe the actions on steady-state current of the inhibitory steroid pregnenolone sulfate. The findings indicate that the steady-state activity in the presence of multiple active agents that interact with distinct binding sites follows standard energetic additivity. The derived equations enable prediction of peak and steady-state activity in the presence of orthosteric and allosteric agonists, and the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: The study describes derivation and properties of a three-state resting-active-desensitized model. The model and associated equations can be used to analyze and predict peak and steady-state activity in the presence of one or more active agents.