Neuronal Inhibition under the Spotlight

In this issue of Neuron, Lin et al. (2015) report an optical method to precisely manipulate the activity of GABAA receptors by designing a mutant receptor that binds photosensitive compounds. This allows for studying GABAA receptors in situ and represents a valuable tool to investigate how inhibition affects brain physiology

Methods for recording and measuring tonic GABAA receptor-mediated inhibition

Tonic inhibitory conductances mediated by GABA(A) receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABA(A) conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current

Snake neurotoxin α-bungarotoxin is an antagonist at native GABAA receptors

AbstractThe snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β–α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus

GABA Potency at GABAA Receptors Found in Synaptic and Extrasynaptic Zones

The potency of GABA is vitally important for its primary role in activating GABAA receptors and acting as an inhibitory neurotransmitter. Although numerous laboratories have presented information, directly or indirectly, on GABA potency, it is often difficult to compare across such studies given the inevitable variations in the methods used, the cell types studied, whether native or recombinant receptors are examined, and their relevance to native synaptic and extrasynaptic GABAA receptors. In this review, we list the most relevant isoforms of synaptic and extrasynaptic GABAA receptors that are thought to assemble in surface membranes of neurons in the central nervous system. Using consistent methodology in one cell type, the potencies of the endogenous neurotransmitter GABA are compared across a spectrum of GABAA receptors. The highest potency for GABA is measured when activating extrasynaptic-type α6 subunit-containing receptors, whereas synaptic-type α2β3γ2 and α3β3γ2 receptors exhibited the lowest potency, and other GABAA receptor subtypes that are found both in synaptic and extrasynaptic compartments, showed intermediate sensitivities to GABA. The relatively simple potency relationship between GABA and its target receptors is important as it serves as one of the major determinants of GABAA receptor activation, with consequences for the development of inhibition, either by tonic or phasic mechanisms

Glycine receptors in GtoPdb v.2023.1

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the GABAA, nicotinic acetylcholine and 5-HT3 receptors and Zn2+- activated channels. The glycine receptor is expressed either as a homo-pentamer of α subunits, or a complex of 4α and 1β subunits [131], that contains an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [20, 84, 94]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the adult form of the receptor contains α1 (or α3) and β subunits whereas the immature form is mostly composed of only α2 subunits [79]. The α4 subunit is a pseudogene in humans [66]. High resolution molecular structures are available for α1 homomeric, α3 homomeric, and αβ hteromeric receptors in a variety of ligand-induced conformations [19, 129, 19, 48, 49, 50]. As in other Cys-loop receptors, the orthosteric binding site for agonists and the competitive antagonist strychnine is formed at the interfaces between the subunits’ extracellular domains. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. This a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [55, 89]. G protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [125, 124]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [97]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [26]. Extracellular Zn2+ potentiates GlyR function at nanomolar concentrations [87]. and causes inhibition at higher micromolar concentrations (17)

Glycine receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors [63]. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [30, 7], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [80, 91, 18]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the mature form of the receptor contains α1 (or α3) and β subunits while the immature form is mostly composed of only α2 subunits. RNA transcripts encoding the α4-subunit have not been detected in adult humans. The N-terminal domain of the α-subunit contains both the agonist and strychnine binding sites that consist of several discontinuous regions of amino acids. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. The latter is a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [86, 51, 53]. G-protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [122, 121]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [94]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [24]

Glycine receptors (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors and Zn2+- activated channels. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [32, 7], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [82, 92, 20]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the adult form of the receptor contains α1 (or α3) and β subunits whereas the immature form is mostly composed of only α2 subunits. The &a;pha;4 subunit is a pseudogene in humans. High resolution molecular structures are available for the α1 and α3 homomeric receptors [49, 19]. As in other Cys-loop receptors, the orthosteric binding site for agonists and the competitive antagonist strychnine is formed at the interfaces between the subunits’ extracellular domains. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. This a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [55, 53, 87]. G protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [123, 122]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [95]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [26]. Extracellular Zn2+ potentiates GlyR function at nanomolar concentrations [85]. and causes inhibition at higher micromolar concentrations (17)

Glycine receptors in GtoPdb v.2021.3

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors and Zn2+- activated channels. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [33, 7], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [83, 93, 21]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the adult form of the receptor contains α1 (or α3) and β subunits whereas the immature form is mostly composed of only α2 subunits. The &a;pha;4 subunit is a pseudogene in humans. High resolution molecular structures are available for the α1 and α3 homomeric receptors [50, 20]. As in other Cys-loop receptors, the orthosteric binding site for agonists and the competitive antagonist strychnine is formed at the interfaces between the subunits’ extracellular domains. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. This a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [56, 54, 88]. G protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [124, 123]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [96]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [27]. Extracellular Zn2+ potentiates GlyR function at nanomolar concentrations [86]. and causes inhibition at higher micromolar concentrations (17)

Glycine receptors in GtoPdb v.2023.1

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the GABAA, nicotinic acetylcholine and 5-HT3 receptors and Zn2+- activated channels. The glycine receptor is expressed either as a homo-pentamer of α subunits, or a complex of 4α and 1β subunits [131], that contains an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [20, 84, 94]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the adult form of the receptor contains α1 (or α3) and β subunits whereas the immature form is mostly composed of only α2 subunits [79]. The α4 subunit is a pseudogene in humans [66]. High resolution molecular structures are available for α1 homomeric, α3 homomeric, and αβ hteromeric receptors in a variety of ligand-induced conformations [19, 129, 19, 48, 49, 50]. As in other Cys-loop receptors, the orthosteric binding site for agonists and the competitive antagonist strychnine is formed at the interfaces between the subunits’ extracellular domains. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. This a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [55, 89]. G protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [125, 124]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [97]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [26]. Extracellular Zn2+ potentiates GlyR function at nanomolar concentrations [87]. and causes inhibition at higher micromolar concentrations (17)

Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress.

BACKGROUND: Bioethanol fermentations follow traditional beverage fermentations where the yeast is exposed to adverse conditions such as oxidative stress. Lignocellulosic bioethanol fermentations involve the conversion of pentose and hexose sugars into ethanol. Environmental stress conditions such as osmotic stress and ethanol stress may affect the fermentation performance; however, oxidative stress as a consequence of metabolic output can also occur. However, the effect of oxidative stress on yeast with pentose utilising capabilities has yet to be investigated. RESULTS: Assaying for the effect of hydrogen peroxide-induced oxidative stress on Candida, Pichia and Scheffersomyces spp. has demonstrated that these yeast tolerate hydrogen peroxide-induced oxidative stress in a manner consistent with that demonstrated by Saccharomyces cerevisiae. Pichia guillermondii appears to be more tolerant to hydrogen peroxide-induced oxidative stress when compared to Candida shehatae, Candida succiphila or Scheffersomyces stipitis. CONCLUSIONS: Sensitivity to hydrogen peroxide-induced oxidative stress increased in the presence of minimal media; however, addition of amino acids and nucleobases was observed to increase tolerance. In particular adenine increased tolerance and methionine reduced tolerance to hydrogen peroxide-induced oxidative stress