Stereospecific Properties and Intracellular Transport of Novel Intrinsically Fluorescent Neurosteroids.

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABA) 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.

Synthesis and evaluation of photoaffinity labeling reagents for identifying binding sites of sulfated neurosteroids on NMDA and GABA receptors.

The endogenous neurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) are allosteric modulators of γ-aminobutyric acid type A (GABA) and -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.

Three classes of propofol binding sites on GABA receptors.

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator of gamma-aminobutyric acid type A (GABA) 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.

Inhibitory Actions of Potentiating Neuroactive Steroids in the Human α1β3γ2L γ-Aminobutyric Acid Type A Receptor.

The γ-aminobutyric acid type A (GABA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3-hydroxy steroids inhibit, while 3-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the 13γ2L GABA receptor by the endogenous neurosteroid 3-hydroxy-5-pregnan-20-one (35P) and the synthetic neuroactive steroid 3-hydroxy-5-androstane-17-carbonitrile (ACN).

Direct measurements of neurosteroid binding to specific sites on GABA receptors.

Background And Purpose: Neurosteroids are allosteric modulators of GABA 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 GABA 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.

Comparison of Behavioral Effects of GABAergic Low- and High-Efficacy Neuroactive Steroids in the Zebrafish Larvae Assay.

Activation of the GABA 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 GABA receptor may act, due to a ceiling effect, as a sedative with reduced and limited action.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

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.

Neurosteroids and their potential as a safer class of general anesthetics.

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.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling. GPR161 mutations lead to developmental defects and cancers. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear.

Neurosteroid enantiomers as potentially novel neurotherapeutics.

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.

The Mechanism of Enantioselective Neurosteroid Actions on GABA Receptors.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABA 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.

Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.

Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified lysosomal cholesterol signaling (LYCHOS, previously annotated as G protein-coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic target of rapamycin complex 1 (mTORC1). Cholesterol bound to the amino-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation.

Neurosteroid Modulation of GABA Receptor Function by Independent Action at Multiple Specific Binding Sites.

Neurosteroids are endogenous modulators of GABA 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 GABA 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.

Perspective on the Relationship between GABAA Receptor Activity and the Apparent Potency of an Inhibitor.

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.

Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone.

Intrasubunit and Intersubunit Steroid Binding Sites Independently and Additively Mediate 122L GABA Receptor Potentiation by the Endogenous Neurosteroid Allopregnanolone.

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABA receptor. The sites are located in the membrane-spanning domains of the receptor at the - subunit interface (site I) and within the (site II) and subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat 122L GABA receptor by the endogenous neurosteroid allopregnanolone (35P).

Protocol for a proof-of-concept observational study evaluating the potential utility and acceptability of a telemedicine solution for the post-anesthesia care unit.

The post-anesthesia care unit (PACU) is a clinical area designated for patients recovering from invasive procedures. There are typically several geographically dispersed PACUs within hospitals. Patients in the PACU can be unstable and at risk for complications.

Reduced Activation of the Synaptic-Type GABA Receptor Following Prolonged Exposure to Low Concentrations of Agonists: Relationship between Tonic Activity and Desensitization.

Synaptic GABA receptors are alternately exposed to short pulses of a high, millimolar concentration of GABA and prolonged periods of low, micromolar concentration of the transmitter. Prior work has indicated that exposure to micromolar concentrations of GABA can both activate the postsynaptic receptors generating sustained low-amplitude current and desensitize the receptors, thereby reducing the peak amplitude of subsequent synaptic response. However, the precise relationship between tonic activation and reduction of peak response is not known.

Site-specific effects of neurosteroids on GABA receptor activation and desensitization.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the αβ GABA receptor (GABAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β(+)-α(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β subunit, promoting receptor desensitization and the α subunit promoting effects that vary between neurosteroids.

Enhancement of Muscimol Binding and Gating by Allosteric Modulators of the GABA Receptor: Relating Occupancy to State Functions.

Muscimol is a psychoactive isoxazole derived from the mushroom and a potent orthosteric agonist of the GABA receptor. The binding of [H]muscimol has been used to evaluate the distribution of GABA receptors in the brain, and studies of modulation of [H]muscimol binding by allosteric GABAergic modulators such as barbiturates and steroid anesthetics have provided insight into the modes of action of these drugs on the GABA receptor. It has, however, not been feasible to directly apply interaction parameters derived from functional studies to describe the binding of muscimol to the receptor.

Analysis of Modulation of the 1 GABA Receptor by Combinations of Inhibitory and Potentiating Neurosteroids Reveals Shared and Distinct Binding Sites.

The 1 GABA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the 1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5-reduced steroids, sulfated or carboxylated steroids, and -estradiol, whereas many 5-reduced steroids potentiate the receptor.

Multiple neurosteroid and cholesterol binding sites in voltage-dependent anion channel-1 determined by photo-affinity labeling.

Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73.