Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.

miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity.

Activity-dependent protein synthesis is crucial for long-lasting forms of synaptic plasticity. However, our understanding of translational mechanisms controlling GABAergic synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the mechanisms controlling plasticity-induced gephyrin translation remain unknown. We identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting inhibitory synaptic structure and function. iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and promoting de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Together, we delineate a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

γ1 GABAA Receptors in Spinal Nociceptive Circuits.

GABAergic neurons and GABAA receptors (GABAARs) are critical elements of almost all neuronal circuits. Most GABAARs of the CNS are heteropentameric ion channels composed of two α, two ß, and one γ subunits. These receptors serve as important drug targets for benzodiazepine (BDZ) site agonists, which potentiate the action of GABA at GABAARs. Most GABAAR classifications rely on the heterogeneity of the α subunit (α1-α6) included in the receptor complex. Heterogeneity of the γ subunits (γ1-γ3), which mediate synaptic clustering of GABAARs and contribute, together with α subunits, to the benzodiazepine (BDZ) binding site, has gained less attention, mainly because γ2 subunits greatly outnumber the other γ subunits in most brain regions. Here, we have investigated a potential role of non-γ2 GABAARs in neural circuits of the spinal dorsal horn, a key site of nociceptive processing. Female and male mice were studied. We demonstrate that besides γ2 subunits, γ1 subunits are significantly expressed in the spinal dorsal horn, especially in its superficial layers. Unlike global γ2 subunit deletion, which is lethal, spinal cord-specific loss of γ2 subunits was well tolerated. GABAAR clustering in the superficial dorsal horn remained largely unaffected and antihyperalgesic actions of HZ-166, a nonsedative BDZ site agonist, were partially retained. Our results thus suggest that the superficial dorsal horn harbors functionally relevant amounts of γ1 subunits that support the synaptic clustering of GABAARs in this site. They further suggest that γ1 containing GABAARs contribute to the spinal control of nociceptive information flow.

Haploinsufficiency of GABAA Receptor-Associated Clptm1 Enhances Phasic and Tonic Inhibitory Neurotransmission, Suppresses Excitatory Synaptic Plasticity, and Impairs Memory.

The mechanisms utilized by neurons to regulate the efficacy of phasic and tonic inhibition and their impacts on synaptic plasticity and behavior are incompletely understood. Cleft lip and palate transmembrane protein 1 (Clptm1) is a membrane-spanning protein that interacts with multiple γ-aminobutyric acid type A receptor (GABAAR) subunits, trapping them in the endoplasmic reticulum and Golgi network. Overexpression and knock-down studies suggest that Clptm1 modulates GABAAR-mediated phasic inhibition and tonic inhibition as well as activity-induced inhibitory synaptic homeostasis in cultured hippocampal neurons. To investigate the role of Clptm1 in the modulation of GABAARs in vivo, we generated Clptm1 knock-out (KO) mice. Here, we show that genetic KO of Clptm1 elevated phasic and tonic inhibitory transmission in both male and female heterozygous mice. Although basal excitatory synaptic transmission was not affected, Clptm1 haploinsufficiency significantly blocked high-frequency stimulation-induced long-term potentiation (LTP) in hippocampal CA3→CA1 synapses. In the hippocampus-dependent contextual fear-conditioning behavior task, both male and female Clptm1 heterozygous KO mice exhibited impairment in contextual fear memory. In addition, LTP and contextual fear memory were rescued by application of L-655,708, a negative allosteric modulator of the extrasynaptic GABAAR α5 subunit. These results suggest that haploinsufficiency of Clptm1 contributes to cognitive deficits through altered synaptic transmission and plasticity by elevation of inhibitory neurotransmission, with tonic inhibition playing a major role.

Correlations of receptor desensitization of gain-of-function GABRB3 variants with clinical severity.

Genetic variants associated with developmental and epileptic encephalopathies have been identified in the GABRB3 gene that encodes the ß3 subunit of GABAA receptors. Typically, variants alter receptor sensitivity to GABA resulting in either gain- or loss-of-function, which correlates with patient phenotypes. However, it is unclear how another important receptor property, desensitization, contributes to the greater clinical severity of gain-of-function variants. Desensitization properties of 20 gain-of-function GABRB3 variant receptors were evaluated using two-electrode voltage-clamp electrophysiology. The parameters measured included current decay rates and steady-state currents. Selected variants with increased or reduced desensitization were also evaluated using whole-cell electrophysiology in transfected mammalian cell lines. Of the 20 gain-of-function variants assessed, 13 were found to alter receptor desensitization properties. Seven variants reduced desensitization at equilibrium, which acts to worsen gain-of-function traits. Six variants accelerated current decay kinetics, which limits gain-of-function traits. All affected patients displayed severe clinical phenotypes with intellectual disability and difficult-to-treat epilepsy. Nevertheless, variants that reduced desensitization at equilibrium were associated with more severe clinical outcomes. This included younger age of first seizure onset (median 0.5 months), movement disorders (dystonia and dyskinesia), epilepsy of infancy with migrating focal seizures (EIMFS) and risk of early mortality. Variants that accelerated current decay kinetics were associated with slightly milder phenotypes with later seizure onset (median 4 months), unclassifiable developmental and epileptic encephalopathies or Lennox-Gastaut syndrome and no movement disorders. Our study reveals that gain-of-function GABRB3 variants can increase or decrease receptor desensitization properties and that there is a correlation with the degree of disease severity. Variants that reduced the desensitization at equilibrium were clustered in the transmembrane regions that constitute the channel pore and correlated with greater disease severity, while variants that accelerated current decay were clustered in the coupling loops responsible for receptor activation and correlated with lesser severity.

Chloride deregulation and GABA depolarization in MTOR related malformations of cortical development.

Focal Cortical Dysplasia, Hemimegalencephaly and Cortical Tuber are pediatric epileptogenic malformations of cortical development (MCDs) frequently pharmaco-resistant and mostly surgically treated by the resection of epileptic cortex. Availability of cortical resection samples allowed significant mechanistic discoveries directly from human material. Causal brain somatic or germline mutations in the AKT/PI3K/DEPDC5/MTOR genes were identified. GABAa mediated paradoxical depolarization, related to altered chloride (Cl-) homeostasis, was shown to participate to ictogenesis in human pediatric MCDs. However, the link between genomic alterations and neuronal hyperexcitability is still unclear. Here we studied the post translational interactions between the mTOR pathway and the regulation of cation-chloride cotransporters (CCC), KCC2 and NKCC1, that are largely responsible for controlling intracellular Cl- and ultimately GABAergic transmission. For this study, 35 children (25 MTORopathies and 10 pseudo controls, diagnosed by histology plus genetic profiling) were operated for drug resistant epilepsy. Postoperative cortical tissues were recorded on multielectrode array (MEA) to map epileptic activities. CCC expression level and phosphorylation status of the WNK1/SPAK-OSR1 pathway was measured during basal conditions and after pharmacological modulation. Direct interactions between mTOR and WNK1 pathway components were investigated by immunoprecipitation. Membranous incorporation of MCD samples in Xenopus laevis oocytes enabled Cl- conductance and equilibrium potential (EGABA) for GABA measurement. Of the 25 clinical cases, half harbored a somatic mutation in the mTOR pathway, while pS6 expression was increased in all MCD samples. Spontaneous interictal discharges were recorded in 65% of the slices. CCC expression was altered in MCDs, with a reduced KCC2/NKCC1 ratio and decreased KCC2 membranous expression. CCC expression was regulated by the WNK1/SPAK-OSR1 kinases through direct phosphorylation of Thr906 on KCC2, that was reversed by WNK1 and SPAK antagonists (NEM and Staurosporine). mSIN1 subunit of MTORC2 was found to interact with SPAK-OSR1 and WNK1. Interactions between these key epileptogenic pathways could be reversed by the mTOR specific antagonist Rapamycin, leading to a dephosphorylation of CCCs and recovery of the KCC2/NKCC1 ratio. The functional effect of such recovery was validated by the restoration of the depolarizing shift in EGABA by rapamycin, measured after incorporation of MCD membranes to X. laevis oocytes, in line with a reestablishment of normal ECl-. Our study deciphers a protein interaction network through a phosphorylation cascade between MTOR and WNK1/SPAK-OSR1 leading to chloride cotransporters deregulation, increased neuronal chloride levels and GABAa dysfunction in malformations of Cortical Development, linking genomic defects and functional effects and paving the way to target epilepsy therapy.

The role of HCN channels on the effects of T-type calcium channels and GABAA receptors in the absence epilepsy model of WAG/Rij rats.

In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for three hours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.

Total Synthesis of (15R)- and (15S)-Prostaglandin A2.

From both pharmaceutical and structural perspectives, the large family of prostaglandins represent a truly remarkable class of natural products. Prostaglandin A2 is a tissue hormone naturally found in human seminal plasma and in the sea whip Plexaura homomalla with yet poorly understood biological or therapeutic effects. Herein, a novel strategy for the stereoselective construction of both naturally occurring prostaglandin A2 epimers and first insights into their functional effects on the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) type A receptors (GABAAR) are provided. The synthesis of both epimers was achieved in only 11 steps starting from commercially available 2,5-dimethoxy-tetrahydrofuran employing an organocatalytic domino-aldol reaction, a Mizoroki-Heck reaction, a Wittig reaction as well as an oxidation-decarboxylation sequence. The (15R)-epimer significantly reduced GABA-induced currents through GABAA receptors while its (15S)-epimer did not show any significant effect. These data suggest that (15R)-PGA2 might serve as a novel scaffold for the development of selective GABAA receptor modulators.

Neurosteroids and translocator protein 18 kDa (TSPO) ligands as novel treatment options in depression.

Recently, the gamma-aminobutyric acid (GABA) system has come into focus for the treatment of anxiety, postpartum depression, and major depressive disorder. Endogenous 3α-reduced steroids such as allopregnanolone are potent positive allosteric modulators of GABAA receptors and have been known for decades. Current industry developments and first approvals by the U.S. food and drug administration (FDA) for the treatment of postpartum depression with exogenous analogues of these steroids represent a major step forward in the field. 3α-reduced steroids target both synaptic and extrasynaptic GABAA receptors, unlike benzodiazepines, which bind to synaptic receptors. The first FDA-approved 3α-reduced steroid for postpartum depression is brexanolone, an intravenous formulation of allopregnanolone. It has been shown to provide rapid relief of depressive symptoms. An orally available 3α-reduced steroid is zuranolone, which also received FDA approval in 2023 for the treatment of postpartum depression. Although a number of studies have been conducted, the efficacy data were not sufficient to achieve approval of zuranolone in major depressive disorder by the FDA in 2023. The most prominent side effects of these 3α-reduced steroids are somnolence, dizziness and headache. In addition to the issue of efficacy, it should be noted that current data limit the use of these compounds to two weeks. An alternative to exogenous 3α-reduced steroids may be the use of substances that induce endogenous neurosteroidogenesis, such as the translocator protein 18 kDa (TSPO) ligand etifoxine. TSPO has been extensively studied for its role in steroidogenesis, in addition to other functions such as anti-inflammatory and neuroregenerative properties. Currently, etifoxine is the only clinically available TSPO ligand in France for the treatment of anxiety disorders. Studies are underway to evaluate its antidepressant potential. Hopefully, neurosteroid research will lead to the development of fast-acting antidepressants.

Differential effects of group III metabotropic glutamate receptors on spontaneous inhibitory synaptic currents in spine-innervating double bouquet and parvalbumin-expressing dendrite-targeting GABAergic interneurons in human neocortex.

Diverse neocortical GABAergic neurons specialize in synaptic targeting and their effects are modulated by presynaptic metabotropic glutamate receptors (mGluRs) suppressing neurotransmitter release in rodents, but their effects in human neocortex are unknown. We tested whether activation of group III mGluRs by L-AP4 changes GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in 2 distinct dendritic spine-innervating GABAergic interneurons recorded in vitro in human neocortex. Calbindin-positive double bouquet cells (DBCs) had columnar "horsetail" axons descending through layers II-V innervating dendritic spines (48%) and shafts, but not somata of pyramidal and nonpyramidal neurons. Parvalbumin-expressing dendrite-targeting cell (PV-DTC) axons extended in all directions innervating dendritic spines (22%), shafts (65%), and somata (13%). As measured, 20% of GABAergic neuropil synapses innervate spines, hence DBCs, but not PV-DTCs, preferentially select spine targets. Group III mGluR activation paradoxically increased the frequency of sIPSCs in DBCs (to median 137% of baseline) but suppressed it in PV-DTCs (median 92%), leaving the amplitude unchanged. The facilitation of sIPSCs in DBCs may result from their unique GABAergic input being disinhibited via network effect. We conclude that dendritic spines receive specialized, diverse GABAergic inputs, and group III mGluRs differentially regulate GABAergic synaptic transmission to distinct GABAergic cell types in human cortex.

Comparison of the negative effect of remimazolam and propofol on cardiac contractility: Analysis of a randomised parallel-group trial and a preclinical ex vivo study.

Remimazolam is a newly developed ultra-short-acting benzodiazepine that exerts sedative effects. This study aimed to clarify the effects of remimazolam on cardiac contractility. In a randomised-parallel group trial, haemodynamic parameters were compared between propofol (n = 11) and remimazolam (n = 12) groups during the induction of general anaesthesia in patients undergoing non-cardiac surgery. In a preclinical study, the direct effects of remimazolam on cardiac contractility were also evaluated using isolated rat hearts. RNA sequence data obtained from rat and human hearts were analysed to assess the expression patterns of the cardiac γ-aminobutyric acid type A (GABAA ) receptor subunits. In a clinical study, the proportional change of the maximum rate of arterial pressure rise was milder during the study period in the remimazolam group (propofol: -52.6 [10.2] (mean [standard deviation])% vs. remimazolam: -39.7% [10.5%], p = 0.007). In a preclinical study, remimazolam did not exert a negative effect on left ventricle developed pressure, whereas propofol did exert a negative effect after bolus administration of a high dose (propofol: -26.9% [3.5%] vs. remimazolam: -1.1 [6.9%], p < 0.001). Analysis of the RNA sequence revealed a lack of γ subunits, which are part of the major benzodiazepine binding site of the GABAA receptor, in rat and human hearts. These results indicate that remimazolam does not have a direct negative effect on cardiac contractility, which might contribute to its milder effect on cardiac contractility during the induction of general anaesthesia. The expression patterns of cardiac GABAA receptor subunits might be associated with the unique pharmacokinetics of benzodiazepines in the heart.

Effect of Lipopolysaccharide on the Duration of Zolpidem-Induced Loss of Righting Reflex in Mice.

Zolpidem, a non-benzodiazepine hypnotic, is primarily used to treat insomnia. In a previous study, pior treatment with non-benzodiazepine receptor agonists was associated with inflammation. The present study aimed to clarify the association between the effects of zolpidem and inflammation in mice treated with lipopolysaccharide (LPS), a known model of inflammation. We assessed the zolpidem-induced loss of righting reflex (LORR) duration 24 h after LPS treatment in mice. Additionally, the expressions of γ-aminobutyric acid (GABA)A receptor subunit and K+-Cl- cotransporter isoform 2 (KCC2) mRNA in the hippocampus and frontal cortex were examined in LPS-treated mice. Pretreatment with LPS was associated with significantly prolonged duration of zolpidem-induced LORR compared to control mice. This effect was significantly attenuated by administering bicuculline, a GABAA receptor antagonist, or flumazenil, a benzodiazepine receptor antagonist, in LPS-treated mice. Compared to controls, LPS-treated mice showed no significant change in the expression of GABAA receptor subunits in the hippocampus or frontal cortex. Bumetanide, an Na+-K+-2Cl- cotransporter isoform 1 blocker, attenuated the extended duration of zolpidem-induced LORR observed in LPS-treated mice. LPS significantly decreased Kcc2 mRNA expression in the hippocampus and the frontal cortex. These findings suggest that inflammation increases zolpidem-induced LORR, possibly through a reduction in KCC2 expression.

Involvement of the GABAA Receptor in the Antidepressant-Like Effects Produced by Low and High Doses of the Flavonoid Chrysin in the Rat: A Longitudinal Study.

BACKGROUND: The flavonoid chrysin produces rapid and long-lasting anxiolytic- and antidepressant-like effects in rats. However, it is not known whether low and high doses of chrysin produce differential anti-immobility effects through the Gamma-Aminobutyric Acid sub-type A (GABAA) receptor. The goal of this work was therefore to compare low and high doses of chrysin for their effects on depression-like behavior in a longitudinal study. Moreover, chrysin was compared with the serotonergic fluoxetine and Gamma-Aminobutyric Acid (GABA)ergic allopregnanolone, and its involvement with the GABAA receptor after chronic treatment was also investigated. METHODS: Male Wistar rats were assigned to five groups (n = 8 each): vehicle, 1 mg/kg chrysin, 5 mg/kg chrysin, 1 mg/kg fluoxetine, and 1 mg/kg allopregnanolone. In the first experiment, treatments were injected daily and the effects on locomotor activity and the forced swim test were evaluated at 0, 1, 14, and 28 days of treatment, and 48 h after the final treatment. In the second experiment, similar groups were treated for 28 days with injection of 1 mg/kg picrotoxin to investigate the role of the GABAA receptor. Depending on the experimental design, one- and two-way analysis of variance (ANOVA) tests were used for statistical analysis, with p < 0.05 set as the criteria for significance. RESULTS: In both experiments, the treatments did not alter locomotor activity. However, low and high doses of chrysin, allopregnanolone, and fluoxetine gradually produced antidepressant-like effects in the forced swim test, and maintained this effect for 48 h post-treatment, except with low dose chrysin. Picrotoxin blocked the antidepressant-like effects produced by low dose chrysin, but did not affect those produced by high dose chrysin, allopregnanolone, or fluoxetine. CONCLUSIONS: The differential antidepressant-like effects caused by low and high doses of chrysin are time-dependent. Low dose chrysin produces a rapid antidepressant-like effect, whereas high dose chrysin produces a delayed but sustained the effect, even 48 h after withdrawal. The effect with high dose chrysin was similar to that observed with allopregnanolone and fluoxetine. The mechanism for the antidepressant-like effect of low chrysin appears to be GABAergic, whereas the effect of high dose chrysin may involve other neurotransmission and neuromodulation systems related to the serotonergic system.

Brain carbon monoxide can suppress the rat micturition reflex through brain γ-aminobutyric acid receptors.

OBJECTIVES: To investigate roles of brain carbon monoxide (CO), an endogenous gasotransmitter, in regulation of the rat micturition reflex. METHODS: In urethane-anesthetized (0.8 g/kg, ip) male rats, evaluation of urodynamic parameters was started 1 h before intracerebroventricular administration of CORM-3 (CO donor) or ZnPP (non-selective inhibitor of heme oxygenase, a CO producing enzyme) and continued for 2 h after the administration. We also investigated effects of centrally pretreated SR95531 (GABAA receptor antagonist) or SCH50911 (GABAB receptor antagonist) on the CORM-3-induced response. RESULTS: CORM-3 significantly prolonged intercontraction intervals (ICIs) without changing maximal voiding pressure (MVP), while ZnPP significantly shortened ICI and reduced single-voided volume and bladder capacity without affecting MVP, post-voided residual volume, or voiding efficiency. The ZnPP-induced ICI shortening was reversed by CORM-3. The CORM-3-induced ICI prolongation was significantly attenuated by centrally pretreated SR95531 or SCH50911, respectively. CONCLUSIONS: Brain CO can suppress the rat micturition reflex through brain γ-aminobutyric acid (GABA) receptors.

Bis-piperidine alkaloids from the peels of Areca catechu.

Four new alkaloids, arecatines A-D (1-4), were isolated from the peels of Areca catechu. Compound 1 is an unusual piperidine-pyridine hybrid alkaloid, whereas compounds 2-4 feature bis-piperidine alkaloids. Their structures were elucidated by UV, IR, HRESIMS, and NMR spectra analysis. The molecular docking analysis indicated that compound 3 exhibited the best binding affinity with the GABAA receptor, indicating its potential anti-epilepsy activity.

Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors.

Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABAA receptors (GABAARs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC50 of 31.5 ± 1.7 and 40.6 ± 8.2 µM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABAARs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS.

Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome.

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.

3,8-Disubstituted Pyrazolo[1,5-a]quinazoline as GABAA Receptor Modulators: Synthesis, Electrophysiological Assays, and Molecular Modelling Studies.

As a continuation of our study in the field of GABAA receptor modulators, we report the design and synthesis of new pyrazolo[1,5-a]quinazoline (PQ) bearing at the 8-position an oxygen or nitrogen function. All the final compounds and some intermediates, showing the three different forms of the pyrazolo[1,5-a]quinazoline scaffold (5-oxo-4,5-dihydro, -4,5-dihydro, and heteroaromatic form), have been screened with an electrophysiological technique on recombinant GABAAR (α1ß2γ2-GABAAR), expressed in Xenopus laevis oocytes, by evaluating the variation in produced chlorine current, and permitting us to identify some interesting compounds (6d, 8a, 8b, and 14) on which further functional assays were performed. Molecular modelling studies (docking, minimization of complex ligand-receptor, and MD model) and a statistical analysis by a Hierarchical Cluster Analysis (HCA) have collocated these ligands in the class corresponding to their pharmacological profile. The HCA results are coherent with the model we recently published (Proximity Frequencies), identifying the residues γThr142 and αHis102 as discriminant for the agonist and antagonist profile.

Modular Structure and Polymerization Status of GABAA Receptors Illustrated with EM Analysis and AlphaFold2 Prediction.

Type-A γ-aminobutyric acid (GABAA) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABAA receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor's distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABAA receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABAA receptor α1, ß2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and ß2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABAA receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits' modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABAA receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases.

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. 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 (GABAA) receptors. The potential advantages of NS analogues as sedative and anesthetic agents will be discussed.