The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Pribilla, I.; Takagi, Tohru; Langosch, Dieter; Bormann, Joachim; Betz, H

doi:10.1002/j.1460-2075.1992.tb05529.x

Cited by 327 publications

(350 citation statements)

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“…The 6Ј residue in the bottom half of the M2 ␣-helices forming the ion channel of the GlyR have been shown to be involved in binding of native ginkgolides as well as picrotoxin and its two components picrotin and picrotoxinin (19,20,25,27,28). The 6Ј residue is highly conserved through the Cys-loop receptor family, being a Thr or a Ser residue in almost all subunits (Fig.…”

Section: The Molecular Basis For Subtype Selectivity Of Ginkgolide X mentioning

confidence: 99%

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

Jensen

Bergmann

Sander

et al. 2010

Journal of Biological Chemistry

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The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and ␥-aminobutyric acid type A receptors (GlyRs and GABA A Rs, respectively) in the fluorescence-based FLIPR TM Membrane Potential assay. The compound inhibited the signaling of all GABA A R subtypes included in the study with high nanomolar/low micromolar IC 50 values, except the 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC 50 values at the homomeric ␣1 and ␣2 GlyRs, whereas it was inactive at the heteromeric ␣1␤ and ␣2␤ subtypes at concentrations up to 300 M. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6 M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6 M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA A R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a ␣1 GlyR homology model revealed two distinct putative binding sites formed by the 6 M2 residues together with the 2 residues and the 10 and 13 residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor. ␥-Aminobutyric acid (GABA)2 and glycine are the predominant inhibitory neurotransmitters in the central nervous system and also maintain important functions in several peripheral tissues (1-5). The ionotropic GABA A and glycine receptors (GABA A Rs and GlyRs) belong to the Cys-loop receptor superfamily, which also comprises nicotinic acetylcholine receptors (nAChRs) and 5-HT 3 receptors (5-HT 3 Rs) (3-9). The Cys-loop receptors are homomeric or heteromeric assemblies of five subunits, and the pentameric receptor complex consists of three domains: an extracellular domain composed of the N-terminal domains of the five subunits, a transmembrane domain formed by the M1-M4 ␣-helices of the five subunits (including an ion channel predominantly formed by the five M2 helices), and an intracellular domain composed primarily of the large second intracellular loops of the five subunits (4, 7). Signal transduction through the Cys-loop receptor is initiated by binding of the agonist to orthosteric sites situated at the interfaces between the N-terminal domains of the subunits, and this elicits a conformation change in the pentameric complex leading to flux of ions through the ion channel. Whereas nAChRs and 5-HT 3

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Section: The Molecular Basis For Subtype Selectivity Of Ginkgolide X mentioning

confidence: 99%

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

Jensen

Bergmann

Sander

et al. 2010

Journal of Biological Chemistry

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“…First, green fluorescent protein expression was used to identify cells expressing GlyR ␤ subunit protein. Second, picrotoxin sensitivity was used as a functional assay of the incorporation of ␤ subunits into heteromeric GlyRs (22). When co-expressed with ␣ subunits in HEK293 cells, incorporation of ␤ subunits increases the picrotoxin IC 50 from around 25 to 500 M in the presence of an EC 50 glycine concentration (23).…”

Section: Mutagenesis and Expression Of Glyr Cdnas-mentioning

confidence: 99%

Insights into the Structural Basis for Zinc Inhibition of the Glycine Receptor

Nevin

Cromer

Haddrill

et al. 2003

Journal of Biological Chemistry

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Histidines 107 and 109 in the glycine receptor (GlyR) ␣ 1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR ␣ 1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His 107 from one subunit and His 109 from an adjacent subunit. This was tested by co-expressing ␣ 1 subunits containing the H107A mutation with ␣ 1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type ␣ 1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent ␣ 1 subunits. No evidence was found for ␤ subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zincbinding sites per ␣ 1 ␤ receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of ␣ 1 homomers. The binding of zinc at the interface between adjacent ␣ 1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

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“…The P subunit is not required for ligand binding (Pribilla et al, 1992) and exists as a single subtype that is widely distributed throughout the CNS during all developmental stages (Malosio et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Enhancement of homomeric glycine receptor function by longchain alcohols and anaesthetics

Mascia

Machu

Harris

1996

British J Pharmacology

191

165

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1 The effects of n-alcohols (ethanol to dodecanol) and anaesthetics on strychnine-sensitive glycine receptors were studied in Xenopus oocytes expressing homomeric al or a2 glycine receptor subunits, with the two electrode voltage-clamp recording technique. 2 The glycine-induced chloride conductance of homomeric a glycine receptors was potentiated by all the alcohols tested when an EC2 concentration of glycine was used. Homomeric al and a2 receptors were potentiated similarly by the n-alcohols, except that low concentrations of ethanol produced greater potentiation with acl, as previously reported. 3 Increasing the n-alcohol carbon number has been shown to increase the potency of the alcohols up to decanol at concentrations corresponding to ECsos for producing loss of righting reflex in tadpoles.However, dodecanol was no more potent than decanol, and only modest potentiation (30-60%) was obtained with dodecanol, in contrast to marked (150-200%) potentiation with the other alcohols. Thus, a 'cut-off' occurred at about dodecanol. 4 Propofol, alphaxalone, pentobarbitone, halothane and enflurane, reversibly potentiated the function of homomeric al glycine receptors at concentrations which represent approximately twice the EC50 for production of anaesthesia in mammals, but ketamine and etomidate were ineffective. 5 Two novel cyclobutane compounds were tested; the anaesthetic compound (1-chloro-1,2,2-trifluorocyclobutane) from 0.5 to 5 mm potentiated the action of glycine in a concentration-dependent manner; however, the non-anaesthetic analogue (1,2-dichloro-hexfluorocyclobutane) had no effect on glycine receptor function at concentrations (25 to 80 YM) predicted to be anaesthetic, based on the lipid solubility of this compound. 6 These results suggest that the a subunits of strychnine-sensitive glycine receptors contain sites of action for n-alcohols, propofol, alphaxalone, pentobarbitone and volatile anaesthetics, but not for ketamine and etomidate. Potentiation of glycine receptor function may contribute to the anaesthetic action of n-alcohols and volatile agents.

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The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Cited by 327 publications

References 33 publications

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

Insights into the Structural Basis for Zinc Inhibition of the Glycine Receptor

Enhancement of homomeric glycine receptor function by longchain alcohols and anaesthetics

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