Human Α1 Glycine Receptor Research Articles

Pulsed Electron Spin Resonance Resolves the Coordination Site of Cu 2+ Ions in α1-Glycine Receptor

Herein, we identify the coordination environment of Cu 2+ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn 2+ and Cu 2+ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn 2+ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu 2+ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu 2+ coordination and stoichiometry. We show that Cu 2+ has one binding site per α1 subunit, and that five Cu 2+ can be coordinated per GlyR. Cu 2+ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu 2+ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn 2+ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.

Transient Currents from Glycine Receptors Depend on Intracellular Chloride

We transfected HEK-293 cells with cDNA for homomeric human α1 glycine receptors. 2-3 days after transfection, we recorded currents and used a submillisecond perfusion system to apply glycine to outside-out patches. The extracellular solution contained 150 mM chloride but the intracellular solution contained either 150 mM chloride (symmetric) or 9 mM chloride and 127 mM acetate (“physiological”). We clamped at a range of potentials near the current reversal potential for each condition. With symmetric chloride, currents were nearly constant during the 70 ms application of 3 mM glycine. With “physiological” chloride, currents reversed direction from inward to outward on the 5 ms time scale during agonist application. Similar biphasic currents were seen with gluconate as the substitute intracellular anion. Biphasic currents were observed at external pH values from 6.4-8.0, with HEPES, TRIS or PIPES as external buffer and with external choline replacing sodium. Activation of GlyR channels is dependent on Clin (J Neurosci 28:11454, 2008) but this cannot explain the observed reversal of currents. We are considering that local changes in Clin can cause this effect or that time-dependent changes in pore selectivity are possible.Support from NIH NS045095 (JPD)View Large Image | View Hi-Res Image | Download PowerPoint Slide

Overexpression and Functional Characterization of the Extracellular Domain of the Human α1 Glycine Receptor

A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.

Hyperekplexia mutation R271L of α1 glycine receptors potentiates allosteric interactions of nortropeines, propofol and glycine with [3H]strychnine binding

Human α1 and hyperekplexia mutant α1(R271L) glycine receptors (GlyRs) were transiently expressed in human embryonic kidney 293 cells for [3H]strychnine binding. Binding parameters were determined using a ternary allosteric model. The hyperekplexia mutation increased the positive cooperativity of 0.3mM propofol and glycine binding by about six times: the cooperativity factor β was 0.26 for α1 GlyRs and 0.04 for α1(R271L) GlyRs. Thus, propofol restored the potency of glycine impaired by the mutation. Five nortropeines, i.e. substituted benzoates of nortropine and a new compound, nortropisetron were prepared and also examined on [3H]strychnine binding. They showed nanomolar displacing potencies amplified by the hyperekplexia mutation. The affinity of nor-O-zatosetron (2.6nM) is one of the highest reported for GlyRs. This binding test offers an in vitro method to evaluate agents against neurological disorders associated with inherited mutations of GlyRs.

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

Pharmacological characterisation of strychnine and brucine analogues at glycine and α7 nicotinic acetylcholine receptors

Strychnine and brucine from the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligand-gated ion channels. In this study, we have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human α1 and α1β glycine receptors and at a chimera consisting of the amino-terminal domain of the α7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT 3A serotonin receptor. Although the majority of the analogues displayed significantly increased K i values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the α1 and α1β subtypes. The structure–activity relationships for the compounds at the α7/5-HT 3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the α7/5-HT 3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure–activity relationships for strychnine and brucine analogues at these ligand-gated ion channels.

Functional characterisation of the human α1 glycine receptor in a fluorescence-based membrane potential assay

In the present study, we have created a stable HEK293 cell line expressing the human homomeric α1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR ® Membrane Potential (FMP) assay, a fluorescence-based high throughput screening assay. In the patch-clamp assay, the α1 GlyR exhibited the properties expected from a strychnine-sensitive glycine-gated chloride channel. In the FMP assay exposure of the cell line to GlyR agonists elicited a concentration-dependent increase in fluorescent intensity, a signal that could be suppressed by pre-incubation with GlyR antagonists. Agonists and antagonists displayed EC 50 and K i values in good agreement with previously reported values from studies of recombinant α1 GlyRs and native α1β GlyRs. The rank orders of potencies was glycine > β-alanine > taurine for the agonists and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the α1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn 2+ inhibited α1 GlyR signalling but in contrast to previous reports the metal ion did not appear to potentiate GlyR function at lower concentrations. Analogously, whereas pregnenolone sulphate inhibited α1 GlyR function, the potentiation of α1 GlyR by pregnenolone in electrophysiological studies could not be reproduced in the assay. In conclusion, the FMP assay may not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand–receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs.

Post-synaptic inhibitory mechanisms of anaesthesia; glycine receptors

(1) The effects on human homomeric α1 glycine receptors of 11 general anaesthetics; four barbiturates, two other intravenous anaesthetics, three volatile anaesthetics and two simple gaseous anaesthetics are described. (2) Pentobarbital and thiopental potentiate the current response to bath applied glycine (50 μM) by 200 and 300%, respectively, at clinically relevant concentrations. (3) Neither methohexital nor phenobarbital had any effect on the current response to bath applied glycine (50 μM). (4) Using maximal doses of applied glycine (1 mM) all the barbiturates acted as non-competitive antagonists. (5) Propofol and etomidate potentiate the current response to bath applied glycine (50 μM) by 200 and 10%, respectively, at clinically relevant concentrations. (6) Etomidate acts as a non-competitive antagonist for doses of glycine above the EC 50 (197 μM). Propofol was without effect using maximal doses of applied glycine (1 mM). (7) Halothane, chloroform and ether potentiated the response to bath applied glycine (50 μM) by 200, 100 and 200%, respectively, at clinically relevant doses. (8) None of the volatile anaesthetics had any effect using maximal doses of applied glycine (1 mM). (9) Nitrous oxide and xenon potentiated the response to bath applied glycine (50 μM) by 75 and 50%, respectively, at clinically relevant doses. Nitrous oxide also potentiated the response using maximal doses of applied glycine (1 mM). (10) These results suggest a role for glycinergic neurotransmission in the production of anaesthesia.

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways.

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [3H]strychnine binding assays. In the wild-type (WT) GlyR, 1 microM zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Glycine activation of human homomeric α1 glycine receptors is sensitive to pressure in the range of the high pressure nervous syndrome

The effect of hyperbaric pressure on the inhibitory glycine receptor has been investigated in voltage-clamped Xenopus oocytes microinjected with cRNA encoding the human α1 glycine receptor subunit. Heterologous expression of the human α1 subunit generated functional glycine-gated channels with properties typical of native receptors. Glycine elicited a concentration-dependent inward current which reversed polarity at −25 mV and was antagonised by nanomolar concentrations of strychnine. Concentration-response curves established for the homomeric α1 glycine receptor at 5, 10 and 15 MPa were progressively shifted to the right with respect to the concentration response curve established at atmospheric pressure (0.1 MPa). Pressure had no effect on the maximal response. The EC 50 values at 0.1, 5, 10 and 15 MPa were 190 μM, 222 μM, 338 μM and 482 μM, respectively. The results demonstrate that a receptor comprised solely of the human α-subunit is sensitive to pressure in the range that affects divers and at which the native rat spinal cord receptor is affected. This finding is discussed in the cortext of the postulated binding sites for glycine and the implications for the design of drugs to protect divers from the effects of pressure.

Pharmacology of gossypol

Strychnine and brucine from the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligand-gated ion channels. In this study, we have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human α1 and α1β glycine receptors and at a chimera consisting of the amino-terminal domain of the α7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT3A serotonin receptor. Although the majority of the analogues displayed significantly increased Ki values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the α1 and α1β subtypes. The structure–activity relationships for the compounds at the α7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the α7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure–activity relationships for strychnine and brucine analogues at these ligand-gated ion channels.