Occupation of Either Site for the Neurosteroid Allopregnanolone Potentiates the Opening of the GABA_A Receptor Induced from Either Transmitter Binding Site

Abstract

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA A receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the ␣ subunits of the receptor, whereas GABA binds to pockets in the interfaces between ␤ and ␣ subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same ␤-␣ pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same ␤-␣ pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the ␣ subunit from the same or the other ␤-␣ pair. We conclude that steroids potentiate the opening of the GABA A receptor induced by either agonist binding site.

Neurosteroid interactions with synaptic and extrasynaptic GABAA receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network excitability

“…Channels of α 4 β 2 δ do not differ in GABA potency from channel pentamers composed of α 4 β 2 without a third subunit type (Mortensen et al, 2012). Moreover, receptors modified to contain a single functional GABA agonist site and/or a single proposed allosteric site for steroidal binding are adequate in eliciting potentiation by allopregnanolone, yielding to the potent sensitivity of neurosteroid activity on receptors (Bracamontes et al, 2011). While this recombinant GABA a receptor study was conducted to disassociate the two known sites for neurosteroid binding, the physiologically existent receptors have much more diverse kinetics relative to direct and allosteric neurosteroid binding, and the details surrounding those binding targets remain unclear.…”

Membrane Progesterone Receptors: Evidence for Neuroprotective, Neurosteroid Signaling and Neuroendocrine Functions in Neuronal Cells

“…1C, 1D). Interestingly, allopregnanolone exerted inhibitory effects on cell death and apoptosis at low physiological concentrations of 100 nM and 20 nM, respectively, whereas modulatory effects of the neurosteroid on GABA A receptor functions are usually observed at μM concentrations [20, 59]. These protective actions of allopregnanolone are likely mediated through the most abundant mPR subtype in GT1-7 cells, mPRα, and by activation of MAPkinase and Akt, as observed previously in teleost granulosa cells and in PR-negative human breast cancer cells [12,45].…”

“…Activation of PRs located in the cytoplasmic compartment can mediate nonclassical progesterone signaling through an interaction with Src kinase [19], but other receptor mechanisms must be invoked to explain the cell surface-initiated progesterone actions identified in many target tissues. Although progesterone and its metabolite, allopregnanolone, have been shown to induce rapid effects in neural tissues through modulation GABA A receptor activity [20, 21], rapid progesterone actions in non-neural tissues, which lack GABA A receptors, must involve alternative receptor mechanisms. There is substantial evidence that a putative membrane receptor, progesterone membrane receptor component one (PGMRC1), is a component of nonclassical signaling by progesterone and other hormones [22–24], but its exact role is uncertain and clear evidence that it functions as a specific progesterone receptor is lacking [22, 25, 26].…”

Benzodiazepines Modulate GABAAReceptors by Regulating the Preactivation Step after GABA Binding

“…However, limiting the disruption to a single specified GABA binding site is difficult when expressing ␣1␤2␥2S heteromeric receptors using separate cDNAs. Previously, concatamers have been combined to constrain receptor subunit position and stoichiometry for GABA A Rs (Minier and Sigel, 2004;Baur et al, 2010;Bracamontes et al, 2011).…”