Activation of the Rat α1β2ε GABAA Receptor by Orthosteric and Allosteric Agonists

Abstract

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 α1β2ε GABAA receptor. We confirm that the α1β2ε receptor exhibits a higher level of constitutive activity than typical 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.

Mutational Analysis of Anesthetic Binding Sites and Their Effects on GABAA Receptor Activation and Modulation by Positive Allosteric Modulators of the α7 Nicotinic Receptor

“…Cysteine-modification experiments were done by exposing a wild-type (control) or a cysteine-mutated receptor to p -chloromercuribenzoic acid (pCMB). Successful modification of a cysteine residue was deduced from irreversible alteration of receptor function following the application of pCMB [ 29 , 30 , 31 ]. The pCMB concentration was 25 µM and the application duration was 30 s. pCMB was applied in the presence of 1 mM GABA, and all drug applications were followed by a 4–5 min wash in ND96.…”

GABA and astrocytic cholesterol determine the lipid environment of GABAAR in cultured cortical neurons

“…Subsequently, another 50µL imaging buffer containing 2mM GABA was added to achieve the final 1mM concentration, followed by drug equilibration. Due to the time needed for the safety latch to reset (~40 seconds), we used 1 mM GABA, which is a high physiological concentration and results in a slow desensitization rate of the receptor [54][55][56][57] .…”