Opioid receptors signal through Gi/o protein and β-arrestin pathways that mediate distinct effects of opiate drugs. While opioid binding and G protein activation are well studied, β-arrestin recruitment remains poorly understood. Here, we determine the complex structure of the kappa opioid receptor (KOR) with β-arrestin1 (βarr1) at 2.60 Å resolution using cryogenic electron microscopy. Structural and mass spectrometry analyses reveal multiple phosphorylation sites and a phospholipid-binding site that specifically enhances arrestin recruitment. The KOR-βarr1 complex adopts a core interaction and exhibits notable differences from other GPCR-βarr1 complexes. Comparisons with the structures of KOR-Nb39 and KOR-Gi1 complexes also reveal distinct structural features in the orthosteric binding site and the KOR-transducer interface that affect signaling bias. Using extensive 3D variation analysis and molecular dynamics simulations, we identify a range of conformational dynamics in both the receptor and βarr1, suggesting an allosteric pathway for arrestin's entry and exit.

In recent years three neuroactive steroids (NAS), brexanolone (allopregnanolone, AlloP), ganaxolone and zuranolone, have been approved for the treatment of neuropsychiatric illnesses including postpartum depression and seizures in a neurodevelopmental syndrome. The approved agents are pregnane steroids and strong positive allosteric modulators (PAMs) of gamma-aminobutyric acid type A receptors (GABAARs). Broad effects on GABAARs play important roles in therapeutic benefits. However, these NAS also have actions on non-GABAR targets that could be important for clinical outcomes. Thus, understanding the broader effects of NAS is potentially important for expanding the therapeutic landscape of these important modulators. The approved NAS as well as other structurally distinct NAS and oxysterols have effects on non-GABAAR receptors and ion channels, along with intracellular actions that could have therapeutic importance, including modulation of cellular stress mechanisms, neuroinflammation, mitochondrial function and autophagy, among others. In this review, we explore GABAergic and other cellular effects of pregnane steroids including novel molecules that have potential therapeutic importance. This work discusses the complex chemical nature of NAS and what is being learned at cellular, molecular, synaptic and brain network levels about key sites of action including GABAARs and other targets.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) ion channels are members of the cyclic nucleotide-binding family and are crucial for regulating cellular automaticity in many excitable cells. HCN channel activation contributes to pain perception, and propofol, a widely used anesthetic, acts as an analgesic by inhibiting the voltage-dependent activity of HCN channels. However, the molecular determinants of propofol action on HCN channels remain unknown. Here, we use a propofol-analog photoaffinity labeling reagent to identify propofol binding sites in the human HCN1 isoform. Mass spectrometry analyses combined with molecular dynamics simulations show that a binding pocket is formed by extracellularly facing residues in the S3 and S4 transmembrane segments in the resting voltage-sensor conformation. Mutations of residues within the putative binding pocket mitigate or eliminate voltage-dependent modulation of HCN1 currents by propofol. Together, these findings reveal a conformation-specific propofol binding site that underlies voltage-dependent inhibition of HCN currents and provides a framework for identifying highly specific modulators of HCN channel gating.

GABAA receptors are inhibitory transmitter-gated ion channels formed of various combinations of the 19 homologous subunits cloned to date. Changes in extracellular pH have been shown to directly activate the receptor or modulate its activity elicited by a chemical agonist, but the direction and magnitude of the effect depend on the subunit composition of the receptor. Here, we investigated the acute effect of protonation on the function of the human β3 homomeric GABAA receptor. We show that the reduction of the pH of the extracellular bath solution directly activates this receptor. H+ concentration-response data yielded a pEC50 of ∼6 (EC50 ∼ 1 μM). The probability of being in the active state ranged from 0.005 at pH 8.5 to 0.37 at saturating [H+]. The activating effect of H+ was rapidly reversible, and H+-activated receptors showed minimal desensitization. Mutation of the conserved histidine residue in the 17' position in the second membrane-spanning helix (H267) to alanine abolished H+-elicited activation, revealing inhibition of constitutive activity at low pH that, in its turn, was removed by additionally introducing the H107G mutation in the extracellular domain. We propose that the previously reported constitutive activity of the β3 receptor at physiological pH reflects H+-elicited activity, expressing the net effect of protonation of H267 and H107 residues.

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. Ortho-propofol diazirine (o-PD) labels β-H267, a pore-lining residue, whereas AziPm labels residues β-M286, β-M227, and α-I239 in the two membrane-facing interfaces [β(+)/α(-) and α(+)/β(-)] between α and β subunits. This study used photoaffinity labeling of α1β3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether β3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects β3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of β3-M286, β3-M227, and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in β3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per β-subunit. The native mass spectrometry data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17' H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1β3 GABAA receptors.

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABAA) receptor. The mechanisms underlying the biological activity of AlloP and other NAS are only partially understood. Here, we present intrinsically fluorescent analogs of AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP) (YX-11), and show, by a combination of spectroscopic and computational studies, that these analogs mimic the membrane properties of AlloP and E-AlloP very well. We found stereospecific differences in the orientation and dynamics of the NAS as well as in their impact on membrane permeability. However, all NAS are unable to condense the lipid bilayer, in stark contrast to cholesterol. Using Förster resonance energy transfer (FRET) and electrophysiological measurements, we show that MQ-323 but not YX-11 binds at the intersubunit site of the ELICα1GABAA receptor and potentiates GABA-induced receptor currents. In aqueous solvents, YX-11 forms aggregates at much lower concentrations than MQ-323, and loading both analogs onto cyclodextrin allows for their uptake by human astrocytes, where they become enriched in lipid droplets (LDs), as shown by quantitative fluorescence microscopy. Trafficking of the NAS analogs is stereospecific, as uptake and lipid droplet targeting is more pronounced for YX-11 compared to MQ-323. In summary, we present novel minimally modified analogs of AlloP and E-AlloP, which enable us to reveal stereospecific membrane properties, allosteric receptor activation, and intracellular transport of these neurosteroids. Our fluorescence design strategy will be very useful for the analysis of other NAS in the future.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric G s . This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for G s coupling. Mutations that prevent sterol binding to GPR161 suppress G s -mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

Background and Purpose: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. Experimental Approach: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-alpha 1GABA(A)R, a chimeric receptor containing transmembrane domains of the alpha(1)-GABA(A) receptor. Tryptophan mutagenesis was used to identify specific interactions. Key Results: Allopregnanolone (3 alpha-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3 beta-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. Conclusions and Implications: The affinity and specificity of neurosteroid binding to two sites in the ELIC-alpha 1GABA(A)R were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

Neurosteroids (NS) are a class of steroids that are synthesized within the central nervous system (CNS). Various NS can either enhance or inhibit CNS excitability and they play important biological roles in brain development, brain function and as mediators of mood. One class of NS, 3α-hydroxy-pregnane steroids such as allopregnanolone (AlloP) or pregnanolone (Preg), inhibits neuronal excitability; these endogenous NS and their analogues have been therapeutically applied as anti-depressants, anti-epileptics and general anesthetics. While NS have many favorable properties as anesthetics (e.g. rapid onset, rapid recovery, minimal cardiorespiratory depression, neuroprotection), they are not currently in clinical use, largely due to problems with formulation. Recent advances in understanding NS mechanisms of action and improved formulations have rekindled interest in development of NS as sedatives and anesthetics. In this review, the synthesis of NS, and their mechanism of action will be reviewed with specific emphasis on their binding sites and actions on γ-aminobutyric acid type A (GABA A ) receptors. The potential advantages of NS analogues as sedative and anesthetic agents will be discussed.

The endogenous neurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) are allosteric modulators of γ-aminobutyric acid type A (GABAA) and N-methyl-d-aspartate (NMDA) type glutamate receptors. Analogues of these endogenous steroid sulfates can be either positive or negative allosteric modulators (PAMs or NAMs, respectively) of these receptors, but there is limited information about the steroid-protein binding interactions that mediate these effects and photoaffinity labeling reagents (PALs) of sulfated steroids have not been reported previously. The synthesis of a panel of ten sulfated steroid analogues containing a diazirine group, five of which also contain an alkyne group for click chemistry reactions, for use in photoaffinity labeling studies to identify binding sites for steroid sulfates that are either positive or negative allosteric modulators is reported. Electrophysiological measurements on cultured rat hippocampal neurons were made to determine the modes of allosteric modulation in comparison to those of PS on both receptors. PALs with the activity profile of PS (NMDA PAM, GABAA NAM) were identified. Unexpectedly, PALs with PAM activity at both receptors were also found. Photolabeling of both receptors by two of the PALs was performed to demonstrate their utility, and by inference those of the other PALs, for future studies to identify binding sites for endogenous steroid sulfates on both receptors.

The g-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3/3- hydroxy steroids inhibit, while 3a-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the a1 /33g2L GABAA receptor by the endogenous neurosteroid 3a-hydroxy-5/3-pregnan-20-one (3a5/3P) and the synthetic neuroactive steroid 3a-hydroxy-5a-androstane-17/3-carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3a5/3P and ACN potentiate the GABAA receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC50s of X13 lM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3a5/3P was sensitive to substitution of the a1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3a5/3P inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the a1 and /33 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3a5/3P and ACN.

Activation of the GABAA receptor is associated with numerous behavioral end points ranging from anxiolysis to deep anesthesia. The specific behavioral effect of a GABAergic compound is considered to correlate with the degree of its functional effect on the receptor. Here, we tested the hypothesis that a low-efficacy allosteric potentiator of the GABAA receptor may act, due to a ceiling effect, as a sedative with reduced and limited action. We synthesized a derivative, named (3α,5β)-20-methyl-pregnane-3,20-diol (KK-235), of the GABAergic neurosteroid 5β-pregnane-3α,20α-diol. Using electrophysiology, we showed that KK-235 is a low-efficacy potentiator of the synaptic-type α1β2γ2L GABAA receptor. In the zebrafish larvae behavioral assay, KK-235 was found to only partially block the inverted photomotor response (PMR) and to weakly reduce swimming behavior, whereas the high-efficacy GABAergic steroid (3α,5α,17β)-3-hydroxyandrostane-17-carbonitrile (ACN) fully blocked PMR and spontaneous swimming. Coapplication of KK-235 reduced the potentiating effect of ACN in an electrophysiological assay and dampened its sedative effect in behavioral experiments. We propose that low-efficacy GABAergic potentiators may be useful as sedatives with limited action.

Endogenous neurosteroids and synthetic neuroactive steroids (NAS) are important targets for therapeutic development in neuropsychiatric disorders. These steroids modulate major signaling systems in the brain and intracellular processes including inflammation, cellular stress and autophagy. In this review, we describe studies performed using unnatural enantiomers of key neurosteroids, which are physiochemically identical to their natural counterparts except for rotation of polarized light. These studies led to insights in how NAS interact with receptors, ion channels and intracellular sites of action. Certain effects of NAS show high enantioselectivity, consistent with actions in chiral environments and likely direct interactions with signaling proteins. Other effects show no enantioselectivity and even reverse enantioselectivity. The spectrum of effects of NAS enantiomers raises the possibility that these agents, once considered only as tools for preclinical studies, have therapeutic potential that complements and in some cases may exceed their natural counterparts. Here we review studies of NAS enantiomers from the perspective of their potential development as novel neurotherapeutics.

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.

Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epiallopregnanolone (3β-OH) are both prevalent in the mammalian brain and produce opposite effects on GABAA receptor function, acting as positive and negative allosteric modulators, respectively. This Perspective provides a model to explain the actions of 3α-OH and 3β-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABAA receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.

BACKGROUND:In electrophysiological experiments, inhibition of a receptor-channel, such as the GABAA receptor, is measured by co-applying an agonist producing a predefined control response with an inhibitor to calculate the fraction of the control response remaining in the presence of the inhibitor. The properties of the inhibitor are determined by fitting the inhibition concentration- response relationship to the Hill equation to estimate the midpoint (IC50) of the inhibition curve Objective: We sought to estimate sensitivity of the fitted IC50 to the level of activity of the control response Methods: The inhibition concentration-response relationships were calculated for models with distinct mechanisms of inhibition. In Model I, the inhibitor acts allosterically to stabilize the resting state of the receptor. In Model II, the inhibitor competes with the agonist for a shared binding site. In Model III, the inhibitor stabilizes the desensitized state.RESULTS:The simulations indicate that the fitted IC50 of the inhibition curve is sensitive to the degree of activity of the control response. In Models I and II, the IC50 of inhibition was increased as the probability of being in the active state (PA) of the control response increased. In Model III, the IC50 of inhibition was reduced at higher PA.CONCLUSION:We infer that the apparent potency of an inhibitor depends on the PA of the control response. While the calculations were carried out using the activation and inhibition properties that are representative of the GABAA receptor, the principles and conclusions apply to a wide variety of receptor- channels.