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Incorporation of the ε subunit into the GABAA receptor has been suggested to confer unusual, but variable, biophysical and pharmacological characteristics to both recombinant and native receptors. Due to their structural similarity with the γ subunits, ε subunits have been assumed to substitute at the single position of the γ subunit in assembled receptors. However, prior work suggests that functional variability in ε-containing receptors may reflect alternative sites of incorporation and of not just one, but possibly multiple ε subunits in the pentameric receptor complex. Here we present data indicating that increased expression of ε, in conjunction with α2 and β3 subunits, results in expression of GABAA receptors with correspondingly altered rectification, deactivation and levels of spontaneous openings, but not increased total current density. We also provide data that the ε subunit, like the β3 subunit, can self-export and data from chimeric receptors suggesting that similarities between the assembly domains of the β3 and the ε subunits may allow the ε subunit to replace the β, as well as the γ, subunit. The substitution of an ε for a β, as well as the γ subunit and formation of receptors with alternative patterns of assembly with respect to ε incorporation may underlie the observed variability in both biophysical and pharmacological properties noted not only in recombinant, but also in native receptors. [ABSTRACT FROM AUTHOR]

GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the ℇ-subunit of the GABAA receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABAA receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant α2β3ℇ-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABAA receptors. The AAS 17α-methyltestosterone (17α-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant α2β3ℇ-receptors, 17α-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17α-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, ∼0.15–0.2) in a concentration-dependent fashion (IC50≈ 9 μm). Kinetic modelling indicated that the inhibition induced by 17α-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant ℇ-subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the ℇ-subunit significantly alters the profile of AAS modulation and that this allosteric inhibition of native GnRH neurons should be considered with regard to AAS disruption of neuroendocrine control. [ABSTRACT FROM AUTHOR]

Abstract: Neural transmission mediated by circuits expressing α2 subunit-containing γ-aminobutyric acid type A (GABAA) receptors is critical for the expression of behaviors known to be altered by anabolic androgenic steroids (AAS). Here we show that micromolar concentrations of AAS, which reflect levels found in steroid abusers, induce positive modulation of currents from α2β3γ2L recombinant receptors elicited by pulses of GABA that mimic synaptic conditions in a manner that is mechanistically distinct from modulation induced at α1β3γ2L receptors. Specifically, at α2-containing receptors, the AAS, 17α-methyltestosterone (17α-MeT) enhanced peak current, slowed deactivation, diminished desensitization, and promoted entry of receptors into more distal states along the activation pathway. Analysis of GABAA receptor-mediated synaptic currents in primary cortical neurons followed by single cell real-time RT-PCR demonstrated that 17α-MeT enhancement of synaptic currents is proportional to the ratio of α2 to α1 subunit mRNA. Finally, we show that the modulation elicited by AAS is not comparable to that produced by micromolar concentrations of other positive allosteric modulators at α2-containing receptors. In sum, these data indicate that AAS elicit effects on GABAA receptor function that depend significantly on α subunit composition and that the mechanism of AAS modulation of GABAA receptors is distinct from that of other positive allosteric modulators. [Copyright &y& Elsevier]