Search

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABA A receptor. The sites are located in the membrane-spanning domains of the receptor at the β - α subunit interface (site I) and within the α (site II) and β subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α 1 β 2 γ 2L GABA A receptor by the endogenous neurosteroid allopregnanolone (3 α 5 α P). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3 α 5 α P, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3 α 5 α P act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ -aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

The ρ 1 GABA A receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ 1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABA A receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5 β -reduced steroids, sulfated or carboxylated steroids, and β -estradiol, whereas many 5 α -reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ 1 GABA A receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3 α 5 β P), 3 α 5 β P sulfate, and β -estradiol. The site for 3 α 5 β P can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT: The study describes modulation of the ρ1 GABA A receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Rationale: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified., Objectives: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors., Results: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific., Conclusions: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

A model of the alignment of neurosteroids and ent-neurosteroids at the same binding site on γ-aminobutyric acid type A (GABAA) receptors was evaluated for its ability to identify the structural features in ent-neurosteroids that enhance their activity as positive allosteric modulators of this receptor. Structural features that were identified included: (1) a ketone group at position C-16, (2) an axial 4α-OMe group, and (3) a C-18 methyl group. Two ent-steroids were identified that were more potent than the anesthetic steroid alphaxalone in their threshold for and duration of loss of the righting reflex in mice. In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50 values similar to those found for allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anesthetic agents and as drugs to treat brain disorders that are ameliorated by positive allosteric modulators of GABAA receptor function.

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 α subunits of the receptor, whereas GABA binds to pockets in the interfaces between β and α 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 β-α 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 β-α 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 α subunit from the same or the other β-α pair. We conclude that steroids potentiate the opening of the GABA(A) receptor induced by either agonist binding site.

Neurosteroids are potent modulators of GABA-A receptors. We have examined the time course of development of potentiation of alpha1beta2gamma2L GABA-A receptors during coapplication of GABA and an endogenous neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP). The simultaneous application of 3alpha5alphaP with 5 microm GABA resulted in a biphasic rising phase of current with time constants of 50-60 ms for the rapid phase and 0.3-3 s for the slow phase. The properties of the rapid phase were similar at all steroid concentrations but the time constant of the slower phase became successively shorter as the steroid concentration was increased. Potentiation developed very rapidly (tau = 130 ms) when cells were preincubated with 300 nm 3alpha5alphaP before application of GABA + 3alpha5alphaP, and in outside-out patch recordings, suggesting that steroid diffusion to intracellular compartments competes with receptor potentiation by depleting the cell membrane of steroid. Very low steroid concentrations (3-5 nm) potentiated GABA responses but the effects took minutes to develop. Intracellular accumulation of a fluorescent steroid analogue followed a similar time course, suggesting that slow potentiation results from slow accumulation within plasma membrane rather than indirect effects, such as activation of second messenger systems. In cell-attached single-channel recordings, where 3alpha5alphaP is normally applied through the pipette solution, addition of steroid to the bath solution dramatically shifted the steroid potentiation concentration-effect curve to lower steroid concentrations. We propose that bath-supplied steroid compensates for the diffusion of pipette-supplied steroid out of the patch to the rest of the cell membrane and/or intracellular compartments. The findings suggest that previous studies overestimate the minimum concentration of steroid capable of potentiating GABA actions at GABA-A receptors. The results have implications for the physiological role of endogenous neurosteroids.

Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the beta configuration at the C2 position of (3alpha,5alpha)-3-hydroxypregnan-20-one (allopregnanolone, 3alpha5alphaP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.

We have examined alpha1beta2gamma2L GABAA receptor modulation by the endogenous steroids allopregnanolone (3alpha5alphaP), pregnenolone sulfate, and beta-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3alpha5alphaP but not that by pregnenolone sulfate or beta-estradiol. One of the three kinetic effects evident in channel potentiation by 3alpha5alphaP, prolongation of the longest-lived open time component (OT3), was affected by ethanol with the midpoint of its dose-response curve moved to lower steroid concentrations by 2 orders of magnitude without significantly affecting the maximal effect. Manipulations designed to affect the ability of 3alpha5alphaP to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the gamma2 subunit, which reduces the ability of 3alpha5alphaP to prolong OT3, also reduces the interaction between ethanol and 3alpha5alphaP. And the presence of the competitive steroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3alpha5alphaP on the GABAA receptor. Together, the findings suggest that steroid interactions with the classic steroid binding site underlie the effect seen in the presence of ethanol, and that ethanol acts by increasing the affinity of 3alpha5alphaP for the site. Tadpole behavioral assays showed that the presence of 3alpha5alphaP at a concentration ineffective at causing changes in tadpole behavior shifted the ethanol dose-response curve for loss of righting reflex to lower concentrations and that this effect was neutralized by coapplication of 17-PA with 3alpha5alphaP.

Neuroactive steroids modulate the function of gamma-aminobutyric acid type A (GABA(A)) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3alpha,5beta)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA(A) receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA(A) receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA(A) receptor function and anesthesia. Photolabeling of 35-kDa protein with [(3)H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA(A) receptor function as evidenced by radioligand ([(35)S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA(A) receptor function normally in VDAC-2-deficient fibroblasts transfected with alpha(1)beta(2)gamma(2) GABA(A) receptor subunits. Finally, the neuroactive steroid pregnanolone [(3alpha,5beta)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1- and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA(A) receptor modulation or anesthesia.

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that ent-ALLO potentiates GABA-elicited currents in α1β3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the β3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the β3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or β3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the β3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG. [ABSTRACT FROM AUTHOR]

Activation of GABAA receptors consisting of α4, β2 (or β3), and δ subunits is a major contributor to tonic inhibition in several brain regions. The goal of this study was to analyze the function of the α4β2δ receptor in the presence of GABA and other endogenous and clinical activators and modulators under steady-state conditions. We show that the receptor has a high constitutive open probability (~0.1), but is only weakly activated by GABA that has a maximal peak open probability (POpen,peak) of 0.4, taurine (maximal POpen,peak = 0.4), or the endogenous steroid allopregnanolone (maximal POpen,peak = 0.2). The intravenous anesthetic propofol is a full agonist (maximal POpen,peak = 0.99). Analysis of currents using a cyclic three-state Resting-Active-Desensitized model indicates that the maximal steady-state open probability of the α4β2δ receptor is ~0.45. Steady-state open probability in the presence of combinations of GABA, taurine, propofol, allopregnanolone and/or the inhibitory steroid pregnenolone sulfate closely matched predicted open probability calculated assuming energetic additivity. The results suggest that the receptor is active in the presence of physiological concentrations of GABA and taurine, but, surprisingly, that receptor activity is only weakly potentiated by propofol. [ABSTRACT FROM AUTHOR]

The synaptic α1β2γ2 GABAA receptor is activated phasically by presynaptically released GABA. The receptor is considered to be inactive between synaptic events when exposed to ambient GABA because of its low resting affinity to the transmitter. We tested the hypothesis that a combination of physiological and/or clinical positive allosteric modulators of the GABAA receptor with ambient GABA generates measurable steady‐state activity. Recombinant α1β2γ2L GABAA receptors were expressed in Xenopus oocytes and activated by combinations of low concentrations of orthosteric (GABA, taurine) and allosteric (the steroid allopregnanolone, the anesthetic propofol) agonists, in the absence and presence of the inhibitory steroid pregnenolone sulfate. Steady‐state activity was analyzed using the three‐state cyclic Resting‐Active‐Desensitized model. We estimate that the steady‐state open probability of the synaptic α1β2γ2L GABAA receptor in the presence of ambient GABA (1 μmol/L), taurine (10 μmol/L), and physiological levels of allopregnanolone (0.01 μmol/L) and pregnenolone sulfate (0.1 μmol/L) is 0.008. Coapplication of a clinical concentration of propofol (1 μmol/L) increases the steady‐state open probability to 0.03. Comparison of total charge transfer for phasic and tonic activity indicates that steady‐state activity can contribute strongly (~20 to >99%) to integrated activity from the synaptic GABAA receptor. [ABSTRACT FROM AUTHOR]