The glycine receptor chloride channel (GlyR), a member of the pentameric Cys-loop ion channel receptor family, mediates inhibitory neurotransmission in the spinal cord, brainstem and retina. They are ...also found presynaptically, where they modulate neurotransmitter release. Functional GlyRs are formed from a total of five subunits (α1–α4, β). Although α subunits efficiently form homomeric GlyRs in recombinant expression systems, homomeric α1, α3 and α4 GlyRs are weakly expressed in adult neurons. In contrast, α2 homomeric GlyRs are abundantly expressed in embryonic neurons, although their numbers decline sharply by adulthood. Numerous lines of biochemical, biophysical, pharmacological and genetic evidence suggest the majority of glycinergic neurotransmission in adults is mediated by heteromeric α1β GlyRs. Immunocytochemical co-localisation experiments suggest the presence of α2β, α3β and α4β GlyRs at synapses in the adult mouse retina. Immunocytochemical and electrophysiological evidence also implicates α3β GlyRs as important mediators of glycinergic inhibitory neurotransmission in nociceptive sensory neuronal circuits in peripheral laminae of the spinal cord dorsal horn. It is yet to be determined why multiple GlyR synaptic subtypes are differentially distributed in these and possibly other locations. The development of pharmacological agents that can discriminate strongly between different β subunit-containing GlyR isoforms will help to address this issue, and thereby provide important insights into a variety of central nervous system functions including retinal signal processing and spinal pain mechanisms. Finally, agents that selectively potentiate different GlyR isoforms may be useful as therapeutic lead compounds for peripheral inflammatory pain and movement disorders such as spasticity.
School of Biomedical Sciences, University of Queensland, Brisbane, Australia
The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ...ion channels. Functional receptors of this family comprise five subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four -subunits ( 1 to 4) and one -subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from 2 to 1 is completed by around postnatal day 20 in the rat. The -subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning GlyR molecular structure and function. Most of the information has been obtained from homomeric 1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs.
Address for reprint requests and other correspondence: J. W. Lynch, School of Biomedical Sciences, Univ. of Queensland, Brisbane QLD 4072, Australia (E-mail: j.lynch{at}uq.edu.au )
Hyperekplexia is a rare neurological disorder characterized by neonatal hypertonia, exaggerated startle responses to unexpected stimuli and a variable incidence of apnoea, intellectual disability and ...delays in speech acquisition. The majority of motor defects are successfully treated by clonazepam. Hyperekplexia is caused by hereditary mutations that disrupt the functioning of inhibitory glycinergic synapses in neuromotor pathways of the spinal cord and brainstem. The human glycine receptor α1 and β subunits, which predominate at these synapses, are the major targets of mutations. International genetic screening programs, that together have analysed several hundred probands, have recently generated a clear picture of genotype-phenotype correlations and the prevalence of different categories of hyperekplexia mutations. Focusing largely on this new information, this review seeks to summarise the effects of mutations on glycine receptor structure and function and how these functional alterations lead to hyperekplexia.
Abstract
We search for an isotropic stochastic gravitational-wave background (GWB) in the 12.5 yr pulsar-timing data set collected by the North American Nanohertz Observatory for Gravitational Waves. ...Our analysis finds strong evidence of a stochastic process, modeled as a power law, with common amplitude and spectral slope across pulsars. Under our fiducial model, the Bayesian posterior of the amplitude for an
f
−2/3
power-law spectrum, expressed as the characteristic GW strain, has median 1.92 × 10
−15
and 5%–95% quantiles of 1.37–2.67 × 10
−15
at a reference frequency of
f
yr
=
1
yr
−
1
;
the Bayes factor in favor of the common-spectrum process versus independent red-noise processes in each pulsar exceeds 10,000. However, we find no statistically significant evidence that this process has quadrupolar spatial correlations, which we would consider necessary to claim a GWB detection consistent with general relativity. We find that the process has neither monopolar nor dipolar correlations, which may arise from, for example, reference clock or solar system ephemeris systematics, respectively. The amplitude posterior has significant support above previously reported upper limits; we explain this in terms of the Bayesian priors assumed for intrinsic pulsar red noise. We examine potential implications for the supermassive black hole binary population under the hypothesis that the signal is indeed astrophysical in nature.
Abstract
We report multiple lines of evidence for a stochastic signal that is correlated among 67 pulsars from the 15 yr pulsar timing data set collected by the North American Nanohertz Observatory ...for Gravitational Waves. The correlations follow the Hellings–Downs pattern expected for a stochastic gravitational-wave background. The presence of such a gravitational-wave background with a power-law spectrum is favored over a model with only independent pulsar noises with a Bayes factor in excess of 10
14
, and this same model is favored over an uncorrelated common power-law spectrum model with Bayes factors of 200–1000, depending on spectral modeling choices. We have built a statistical background distribution for the latter Bayes factors using a method that removes interpulsar correlations from our data set, finding
p
= 10
−3
(≈3
σ
) for the observed Bayes factors in the null no-correlation scenario. A frequentist test statistic built directly as a weighted sum of interpulsar correlations yields
p
= 5 × 10
−5
to 1.9 × 10
−4
(≈3.5
σ
–4
σ
). Assuming a fiducial
f
−2/3
characteristic strain spectrum, as appropriate for an ensemble of binary supermassive black hole inspirals, the strain amplitude is
2.4
−
0.6
+
0.7
×
10
−
15
(median + 90% credible interval) at a reference frequency of 1 yr
−1
. The inferred gravitational-wave background amplitude and spectrum are consistent with astrophysical expectations for a signal from a population of supermassive black hole binaries, although more exotic cosmological and astrophysical sources cannot be excluded. The observation of Hellings–Downs correlations points to the gravitational-wave origin of this signal.
BACKGROUND AND PURPOSE Docking studies predict that the insecticides, lindane and fipronil, block GABAA receptors by binding to 6′ pore‐lining residues. However, this has never been tested at any ...Cys‐loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABAA receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1β, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors.
EXPERIMENTAL APPROACH Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch‐clamp electrophysiology.
KEY RESULTS Both compounds completely inhibited all tested glycine receptor subtypes with IC50 values ranging from 0.2–2 µM, similar to their potencies at vertebrate GABAA receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6′ threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2′ level. We present evidence for fipronil binding in a non‐blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of β subunits in αβ heteromeric glycine receptors.
CONCLUSIONS AND IMPLICATIONS This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore‐lining 6′ threonine residues, whereas fipronil may have both pore and non‐pore binding sites.
Ivermectin is a gold standard antiparasitic drug that has been used successfully to treat billions of humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had ...been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular modelling now explain how ivermectin binds to these receptors and reveal why it is selective for invertebrate members of the Cys-loop receptor family. Combining this with emerging genomic information, we are now in a position to predict species sensitivity to ivermectin and better understand the molecular basis of ivermectin resistance. An understanding of the molecular structure of the ivermectin binding site, which is formed at the interface of two adjacent subunits in the transmembrane domain of the receptor, should also aid the development of new lead compounds both as anthelmintics and as therapies for a wide variety of human neurological disorders.
Abstract
The NANOGrav 15 yr data set shows evidence for the presence of a low-frequency gravitational-wave background (GWB). While many physical processes can source such low-frequency gravitational ...waves, here we analyze the signal as coming from a population of supermassive black hole (SMBH) binaries distributed throughout the Universe. We show that astrophysically motivated models of SMBH binary populations are able to reproduce both the amplitude and shape of the observed low-frequency gravitational-wave spectrum. While multiple model variations are able to reproduce the GWB spectrum at our current measurement precision, our results highlight the importance of accurately modeling binary evolution for producing realistic GWB spectra. Additionally, while reasonable parameters are able to reproduce the 15 yr observations, the implied GWB amplitude necessitates either a large number of parameters to be at the edges of expected values or a small number of parameters to be notably different from standard expectations. While we are not yet able to definitively establish the origin of the inferred GWB signal, the consistency of the signal with astrophysical expectations offers a tantalizing prospect for confirming that SMBH binaries are able to form, reach subparsec separations, and eventually coalesce. As the significance grows over time, higher-order features of the GWB spectrum will definitively determine the nature of the GWB and allow for novel constraints on SMBH populations.
Mutations in synaptic NMDA receptors (NMDARs) are associated with epilepsy and neurodevelopmental disorders. The effects of several such mutations have been investigated in recombinantly-expressed ...NMDARs under conditions of steady-state activation. Such experiments provide only limited insight into how mutations affect NMDAR-mediated excitatory synaptic currents (EPSCs). The present study aimed to characterize the effects of the GluN2AN615K, GluN2BN615I and GluN2BV618G gain-of-function mutations on EPSCs mediated by diheteromeric GluN1/2A and GluN1/2B receptors and triheteromeric GluN1/2A/2B receptors, as these are the most abundant synaptic NMDARs in vivo. Subunit composition was controlled by studying ‘artificial’ synapses formed between cultured neurons (which provide presynaptic terminals) and HEK293 cells that express the NMDAR subunits of interest plus the synapse-promoting molecule, neuroligin-1B. When incorporated into diheteromeric receptors, all three mutations ablated voltage-dependent Mg2+ block of EPSCs, as previously shown. In addition, we were surprised to find that increasing external Mg2+ from 0 to 1 mM strongly enhanced the magnitude of EPSCs mediated by mutant diheteromers. In contrast, triheteromeric receptors exhibited normal voltage-dependent Mg2+ block. The GluN2AN615K mutation also slowed the decay of GluN1/2A/2B- but not GluN1/2A-mediated EPSCs. The GluN2BN615I mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs. The GluN2BV618G mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs, although these effects were partly compensated by a faster EPSC decay rate. The mutations also diminished the potency of the anti-epileptic pore-blocker, memantine, thus explaining the lack of memantine efficacy in patients with GluN2BN615I or GluN2BV618G mutations. Given these effects, the three mutations would be expected to enhance the cation influx rate and thereby contribute to epilepsy phenotypes.
•All three mutations eliminated voltage-dependent Mg2+ block of diheteromeric EPSCs•Increasing Mg2+ from 0 to 1 mM enhanced the size of mutant diheteromeric EPSCs•Mutant triheteromeric EPSCs exhibited normal voltage-dependent Mg2+ block•They also exhibited larger magnitudes and/or altered decay rates•These mutations would enhance neuronal excitability, promoting epilepsy phenotypes
Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and ...pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique.
This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.
•Voltage clamp fluorometry reports ion channel conformational changes in real time.•It thus defines domains involved in activation, modulation and desensitization.•It is particularly important for resolving drug modulatory mechanisms.•The incorporation of fluorescent unnatural amino acids will enhance its utility.
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