Pregnenolone sulfate analogues differentially modulate GABAA receptor closed/desensitised states

BACKGROUND AND PURPOSE: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. EXPERIMENTAL APPROACH: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. KEY RESULTS: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. CONCLUSIONS AND IMPLICATIONS: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABAA receptor drug design and development

Functional characterization of GABAA receptor-mediated modulation of cortical neuron network activity in microelectrode array recordings

The numerous γ-aminobutyric acid type A receptor (GABAAR) subtypes are differentially expressed and mediate distinct functions at neuronal level. In this study we have investigated GABAAR-mediated modulation of the spontaneous activity patterns of primary neuronal networks from murine frontal cortex by characterizing the effects induced by a wide selection of pharmacological tools at a plethora of activity parameters in microelectrode array (MEA) recordings. The basic characteristics of the primary cortical neurons used in the recordings were studied in some detail, and the expression levels of various GABAAR subunits were investigated by western blotting and RT-qPCR. In the MEA recordings, the pan-GABAAR agonist muscimol and the GABABR agonist baclofen were observed to mediate phenotypically distinct changes in cortical network activity. Selective augmentation of αβγ GABAAR signaling by diazepam and of δ-containing GABAAR (δ-GABAAR) signaling by DS1 produced pronounced changes in the majority of the activity parameters, both drugs mediating similar patterns of activity changes as muscimol. The apparent importance of δ-GABAAR signaling for network activity was largely corroborated by the effects induced by the functionally selective δ-GABAAR agonists THIP and Thio-THIP, whereas the δ-GABAAR selective potentiator DS2 only mediated modest effects on network activity, even when co-applied with low THIP concentrations. Interestingly, diazepam exhibited dramatically right-shifted concentration-response relationships at many of the activity parameters when co-applied with a trace concentration of DS1 compared to when applied alone. In contrast, the potencies and efficacies displayed by DS1 at the networks were not substantially altered by the concomitant presence of diazepam. In conclusion, the holistic nature of the information extractable from the MEA recordings offers interesting insights into the contributions of various GABAAR subtypes/subgroups to cortical network activity and the putative functional interplay between these receptors in these neurons