Null method to estimate the maximal PA at subsaturating concentrations of agonist.

The maximal probability of being in an active state (PA,max) is a measure of gating efficacy for a given agonist acting on a given receptor channel. In macroscopic electrophysiological recordings, PA,max is typically estimated by comparing the amplitude of the current response to a saturating concentration of a test agonist to that of a reference agonist with known PA. Here, we describe an approach to estimate the PA,max for low-efficacy agonists at subsaturating concentrations.

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.

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.

Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABA receptor and Ca2.2 channel.

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1β2γ2L γ-aminobutyric acid type A receptor (GABAR), and the human voltage-gated N-type calcium channel (Ca2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3β2 ≅ α3β4, indicating that β2/β4 subunits are relatively less important for their activity.

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.

UNC-49 is a redox-sensitive GABA receptor that regulates the mitochondrial unfolded protein response cell nonautonomously.

The γ-aminobutyric acid-mediated (GABAergic) system participates in many aspects of organismal physiology and disease, including proteostasis, neuronal dysfunction, and life-span extension. Many of these phenotypes are also regulated by reactive oxygen species (ROS), but the redox mechanisms linking the GABAergic system to these phenotypes are not well defined. Here, we report that GABAergic redox signaling cell nonautonomously activates many stress response pathways in and enhances vulnerability to proteostasis disease in the absence of oxidative stress.

Potentiation of the GABAR reveals variable energetic contributions by etiocholanolone and propofol.

The properties of a potentiator are typically evaluated by measuring its ability to enhance the magnitude of the control response. Analysis of the ability of drugs to potentiate responses from receptor channels takes place in the context of particular models to extract parameters for functional effects. In the often-used coagonist model, the agonist generating control activity and the potentiator enhancing the control activity make additive energetic contributions to stabilize the active state of the receptor.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABA and the nicotinic 7 receptor modulator (E)-3-furan-2-yl--p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary GABA receptor.

Mutational Analysis of Anesthetic Binding Sites and Their Effects on GABA Receptor Activation and Modulation by Positive Allosteric Modulators of the α7 Nicotinic Receptor.

The positive allosteric modulators (PAMs) of the α7 nicotinic receptor -(5-Cl-2-hydroxyphenyl)-'-[2-Cl-5-(trifluoromethyl)phenyl]-urea (NS-1738) and ()-3-(furan-2-yl)--(-tolyl)-acrylamide (PAM-2) potentiate the α1β2γ2L GABA receptor through interactions with the classic anesthetic binding sites located at intersubunit interfaces in the transmembrane domain of the receptor. In the present study, we employed mutational analysis to investigate in detail the involvement and contributions made by the individual intersubunit interfaces to receptor modulation by NS-1738 and PAM-2. We show that mutations to each of the anesthetic-binding intersubunit interfaces (β+/α-, α+/β-, and γ+/β-), as well as the orphan α+/γ- interface, modify receptor potentiation by NS-1738 and PAM-2.

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.

Activation of the Rat α1β2ε GABA Receptor by Orthosteric and Allosteric Agonists.

GABA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABA subunits cloned to date.

(+)-Catharanthine potentiates the GABA receptor by binding to a transmembrane site at the β(+)/α(-) interface near the TM2-TM3 loop.

(+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABAR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABAR subtype selectivity and location of the putative binding site for (+)-catharanthine using electrophysiological, site-directed mutagenesis, functional competition, and molecular docking experiments. Electrophysiological and in silico experiments showed that (+)-catharanthine potentiates the responses to low, subsaturating GABA at β2/3-containing GABARs 2.

Activation of the α1β2γ2L GABA Receptor by Physiological Agonists.

The Cl permeable GABA receptor is a major contributor to cellular inhibition in the brain. The receptor is normally activated by synaptically-released or ambient GABA but is sensitive to a number of physiological compounds such as β-alanine, taurine, and neurosteroids that, to various degrees, activate the receptor and modulate responses either to the transmitter or to each other. Here, we describe α1β2γ2L GABA receptor activation and modulation by combinations of orthosteric and allosteric activators.

Assessing potentiation of the (α4)3(β2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI.

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity "noncanonical" ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination.

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.

The Sulfated Steroids Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate Inhibit the 132L GABA Receptor by Stabilizing a Novel Nonconducting State.

The GABA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human 132L GABA receptor.

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.

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).

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.