Date Published: January 23, 2017
Publisher: Public Library of Science
Author(s): Roland Baur, Erwin Sigel, Israel Silman.
The major subunit isoform of GABAA receptors is α1β2γ2. The subunits are thought to surround an ion pore with the counterclockwise arrangement α1γ2β2α1β2 as seen from the outside of the neuron. These receptors have two agonist sites and one high affinity drug binding site specific for benzodiazepines. Recently, this receptor was postulated to assume alternative subunit stoichiometries and arrangements resulting in only one agonist site and one or even two sites for benzodiazepines. In order to force a defined subunit arrangement we expressed a combination of triple and dual concatenated subunits. Here we report that these unconventional receptors express only small current amplitudes in Xenopus oocytes. We determined agonist properties and modulation by diazepam of two of these receptors that resulted in currents large enough for a characterization, that is, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2. The first pentamer predicted to have two benzodiazepine binding sites shows similar response to diazepam as the standard receptor. As expected for both receptors with a single predicted agonist site the concentration response curves for GABA were characterized by a Hill coefficient < 1. β2-α1-γ2/β2-γ2 displayed a mM apparent GABA affinity for channel opening instead of the expected μM affinity. Based on their subunit and binding site stoichiometry, that contradicts all previous observations, their unusual functional properties and their very low expression levels in oocytes, we consider it unlikely that these unconventional receptors are expressed in neurons to an appreciable extent.
GABAA receptors are the major inhibitory neurotransmitter receptors in mammalian brain [1–3]. The major isoform of this receptor is composed of α1, β2 and γ2 subunits. The consensus subunit stoichiometry is 2α1, 2β2 and 1γ2 [4,5], arranged α1γ2β2α1β2 counterclockwise as seen from the synaptic cleft [6–9]. α1β2γ2 receptors have one benzodiazepine binding site and two GABA sites , located at the α1/γ2 and β2/α1 subunit interfaces, respectively in a homologous position . The presence of more than one agonist site for GABA is also predicted from their GABA concentration dependence that are characterized by a Hill coefficient of >1 (e.g. ). These α1β2γ2 receptors, unlike many other isoform containing δ or ε subunits [11–15], are thought to have a well-defined subunit arrangement. Subunit stoichiometry and arrangement are important as they govern functional properties . Recently, unconventional subunit arrangements of α1β2γ2 receptors have been proposed . Some of these receptors do not conform to the above stoichiometry as they contain 2γ2 subunits. Furthermore, these receptors are predicted to have one agonist site only and some of them two benzodiazepine sites. Functional properties of these novel receptors, such as EC50 and corresponding Hill coefficient for the agonist GABA and modulation by diazepam were not analyzed. We set out to determine these properties. While receptors conforming the conventional subunit arrangement could easily expressed in Xenopus oocytes, the receptors with unconventional arrangement form rather inefficiently, making their analysis difficult. Nevertheless, we describe the properties of some of them, but think that if these receptors are formed at all, they represent only a very minor part of α1β2γ2 GABAA receptors.
The subunit arrangement of the α1β2γ2 GABAA receptor has been derived from subunit interface studies  and from work with concatenated subunits [7–9]. Fig 1 shows a this arrangement characterized by the presence of two β2/α1 subunit interfaces each harboring agonist sites for GABA and one α1/γ2 subunit interface harboring a benzodiazepine binding site. This is in agreement with the observation that receptors purified from bovine brain have more than one GABA site per benzodiazepine binding site . GABA concentration response curves obtained for α1β2γ2 GABAA receptors also indicate the presence of more than one agonist site (e.g. ), as their Hill coefficient is > 1. In addition to the consensus receptor, Fig 1 shows three unconventional receptors  with one predicted agonist site each, two of them with one, one of them with two predicted benzodiazepine binding sites. All these unconventional receptors do not conform to the well-established subunit stoichiometry of 2:2:1 for α1, β2 and γ2 subunits [4,5]. We attempted to characterize the functional properties of these unconventional receptors.