Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1β2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement

Abstract

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.

Concatenated γ-aminobutyric acid type A receptors revisited: Finding order in chaos

“…This was not reported originally, although careful examination of the data in Baumann et al (2001) reveals low levels of dimer-only expression. Later publications from the Sigel group also note the existence of functional receptors from expression of dimeric constructs by themselves (Kaur et al, 2009; Sigel et al, 2009; Baur and Sigel, 2017). Nevertheless, the key point is that dimers from both constructs assembled readily into fully functional receptors, likely with dangling subunits (Fig.…”

SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

“…Here we employed a ratio of 1:1:4 to minimise the occurrence of diheteromeric receptors. On the other hand, it has been shown that overexpression of γ2 subunits may result in an increased occurrence of receptors containing two γ2 subunits (Quirk et al, 1994 ; Benke et al, 1996 ; Botzolakis et al, 2016 ; Baur and Sigel, 2017 ). Incorporation of a second γ2 subunit (to produce α1γ2α1γ2β2 receptors) has been shown to cause a dramatic slowing in the GABAergic current deactivation rate relative to both the standard diheteromeric and triheteromeric GABA A R isoforms (i.e., α1β2α1β2β2 or α1β2α1γ2β2 receptors; Botzolakis et al, 2016 ).…”