Modulation Of Glycine Receptors Research Articles

Molecular Sites for the Positive Allosteric Modulation of Glycine Receptors by Endocannabinoids

Glycine receptors (GlyRs) are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs) have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA) are positive modulators of α1, α2 and α3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly) potentiate α1 GlyRs but inhibit α2 and α3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM) region 2 and intracellular lysine 385 determine the positive modulation of α1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α1 GlyRs, without affecting inhibition of α2 and α3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/β-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Frequency-Dependent Cannabinoid Receptor-Independent Modulation of Glycine Receptors by Endocannabinoid 2-AG

Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1–1 μM), 2-AG directly affected the functions of recombinant homomeric α1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ∼300 ms. Addition of 1 μM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4–10 Hz) application of short (2 ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10–20 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner.

Molecular Requirements for Ethanol Differential Allosteric Modulation of Glycine Receptors Based on Selective Gβγ Modulation

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult α(1) and embryonic α(2) subunits, can be modified through selective mutations that rescued or impaired Gβγ modulation. Even though both isoforms were able to physically interact with Gβγ, only the α(1) GlyR was functionally modulated by Gβγ and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in α(2) GlyRs was enough to generate GlyRs modulated by Gβγ and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective Gβγ modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.

Ircinialactams: Subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure–activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against α1 and α3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.

Discovering new glycine receptor modulators and their binding sites

The inhibitory glycine receptor (GlyR) is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific modulators. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384-well plate using nanogram quantities of plasmid DNA.

A Transmembrane Residue Influences the Interaction of Propofol with the Strychnine-Sensitive Glycine α1 and α1β Receptor

Propofol, well known for its anesthetic effects, acts as a positive allosteric modulator of the alpha-aminobutyric acid type A (GABA(A)) receptor but also enhances the function of the glycine receptor. The GABA modulatory effects of propofol are influenced by an amino acid residue located within the second transmembrane domain (TM2) of the GABA(A) receptor beta subunit. In glycine alpha(1) subunits, the homologous residue (serine 267) affects the glycine modulatory actions of alcohols and alkane anesthetics. In the present study we investigated the role of this residue on the interaction of propofol with the glycine alpha(1) and alpha(1)beta receptor. The influence of propofol on wild type and mutant (alpha(1)S267M, alpha(1)S267I, alpha(1)S267Mbeta, alpha(1)S267Ibeta) glycine receptors expressed in human embryonic kidney 293 cells was investigated by using the whole-cell clamp technique. Mutation of the alpha(1) subunit TM2 serine residue to either isoleucine or methionine decreased the sensitivity of the receptor to glycine, and abolished the direct activation of the glycine receptor by propofol. Additionally, the methionine and particularly the isoleucine mutation decreased the glycine-enhancing actions of propofol. The nature of the TM2 residue (267) of the glycine alpha(1) subunit influences the glycine modulatory effect of propofol and direct activation of the receptor by this anesthetic. A comparison of the impact of such complementary mutations on the interaction of propofol with glycine and GABA(A) receptors should permit a better understanding of the molecular determinants of action of propofol on these structurally related receptors and may aid in the development of selective glycine receptor modulators.

Subunit-specific modulation of glycine receptors by cannabinoids and N-arachidonyl-glycine

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord motor and pain sensory neurons. Recent studies demonstrated apparently contradictory (potentiating versus inhibitory) effects of the endocannabinoid anandamide on these receptors. The present study characterised the effects of cannabinoid agonists on α1, α1β, α2 and α3 GlyRs recombinantly expressed in HEK293 cells with the aims of reconciling effects of cannabinoids on these receptor subtypes and to establish the potential of different GlyR isoforms as novel physiological or analgesic targets for cannabinoids. The compounds investigated were anandamide, HU-210, HU-308, WIN55,212-2 and the endogenous non-cannabinoid, N-arachidonyl-glycine. The latter compound was chosen due to the structural similarity with anandamide and known analgesic actions in the spinal cord. Recombinant α1 and α1β GlyRs were potentiated by anandamide and HU-210 at submicromolar concentrations, whereas WIN55,212-2 had no effect and HU-308 produced only weak inhibition. By contrast, N-arachidonyl-glycine exerted complex effects including both potentiation and inhibition. Anandamide had no effect at α2 or α3 GlyRs although the other cannabinoids produced potent inhibition. On α2 GlyRs, the inhibitory potency sequence was HU-210 = WIN55,212-2 > HU-308 > N-arachidonyl-glycine but on α3 GlyRs it was HU-210 = WIN55212 = HU-308 > N-arachidonyl-glycine. These results suggest that α1, α2 and α3 containing GlyRs exhibit distinct pharmacological profiles for cannabinoids. We conclude that cannabinoid agonists may be useful as pharmacological tools for selectively inhibiting α2 and α3 GlyRs. Our results also establish GlyRs as potential novel targets for endogenous and exogenous cannabinoids.

Zinc modulation of glycine receptors in acutely isolated rat CA3 neurons

Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippocampus, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch clamp techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl − currents in a concentration-dependent manner. The glycine-induced current ( I Gly) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01–1 µM), ICS-205,930 potentiated I Gly, but at higher concentrations (> 10 µM) it inhibited I Gly. These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing α2 subunits. Furthermore, at lower concentrations (1–30 µM), Zn 2+ potentiated I Gly, but at higher concentrations (> 100 µM) it inhibited I Gly. Considering that Zn 2+ is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn 2+ modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.

Identification of residues mediating inhibition of glycine receptors by protons

We previously identified H109 of the glycine α1 subunit as a putative proton binding site. In the present studies, we explored additional proton binding site(s) as well as the mechanism underlying modulation of glycine receptors by protons. Whole-cell glycine currents were recorded from HEK 293 cells transiently expressing wild type or mutant glycine receptors. Individual mutation of 3 of 4 remaining extracellular histidine residue into alanine (i.e., α1 H107A, H215A or H419A), reduced the receptor sensitivity to protons to a varying extent. In contrast, mutation of α1 H201A did not affect proton sensitivity. Double, triple or quadruple histidine mutation of these residues caused a further reduction of proton sensitivity, suggesting multiple binding sites for proton action on glycine receptors. Furthermore, the substitution T133A, which mediates Zn 2+ inhibition, virtually abolished the proton effect on peak amplitude and current kinetics of glycine response. Replacement of T with S on position 133 partially restored receptor sensitivity to protons, suggesting the hydroxyl group of residue T133 is essential for proton-mediated modulation. In heteromeric α1β receptors, mutations β H132A and S156A, which correspond to H109 and T133 of the α1 subunit, respectively, also affected proton inhibition. In conclusion, multiple extracellular histidine residues (H107, H109, H215 and H419) and threonine residues of the α1 and β Zn 2+ coordination sites are critical for modulation of the glycine receptor by protons.

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.

Modulation of glycine receptors by phenol derivatives

Modulation of glycine receptors by phenol derivatives

Synthesis of Tropeines and Allosteric Modulation of Ionotropic Glycine Receptors

Twenty esters of 3 alpha- and 3beta-hydroxy(nor)tropanes and two amides of 3 alpha-aminotropane were prepared with substituted benzoic acids. These (nor)tropeines inhibited [(3)H]strychnine binding to glycine receptors in synaptosomal membranes of rat spinal cord. A ternary allosteric model was applied to determine the dissociation constants (K(A)) of the tropeines having strong negative cooperativities with [(3)H]strychnine binding (alpha > 10). K(A) values about 10 nM are well below those of known allosteric agents. Low concentrations (0.1K(A)) of the (nor)tropeines potentiated the displacing effects of glycine. Positive cooperativity with glycine (beta < 1) decreased with the increase in concentration and binding affinity of tropeines. Displacing potencies were also measured for [(3)H]granisetron binding to 5-HT(3) type serotonin receptors of rat cerebral cortex. Selectivities to glycine receptors versus 5-HT(3) receptors varied within 4 orders of magnitude. Nortropeines might serve as a lead to high-affinity selective allosteric modulators of glycine receptors.

Molecular Determinants of Proton Modulation of Glycine Receptors

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.

Allosteric modulation of glycine receptors is more efficacious for partial rather than full agonists

Allosteric modulation of [ 3 H ]strychnine binding to glycine receptors (GlyRs) was examined in synaptosomal membranes of rat spinal cord. An allosteric model enabled us to determine the cooperativity factors of the allosteric agents with [ 3 H ]strychnine and glycine bindings (α and β, respectively). We modified the allosteric model with a slope factor because the slope values of the displacement curves of partial agonists (β-alanine, taurine and γ-aminobutyric acid) were beyond unity. The slope factor was reduced only by 100 μM propofol. Further, propofol showed positive cooperativity ( β<1) stronger with taurine than with glycine. The extent of the positive cooperativity of propofol was nearly independent from the potencies and structures of partial agonists. The steroidal alphaxalone and minaxolone also potentiated taurine better than glycine. Alphaxalone exerted weak negative cooperativity with [ 3 H ]strychnine binding. Displacement by taurine is attenuated by granisetron and m-chlorophenylbiguanide representing negative cooperativity ( β⪢1) greater than with glycine. The results suggest a developmental role of elevated perinatal levels of taurine and neurosteroids as well as a better allosteric modulation of decreased agonist efficacies for impaired glycine receptor-ionophores.

Subunit-specific modulation of glycine receptors by neurosteroids

The effects of pregnene and androstane steroids were studied on recombinant human glycine receptors (GlyRs) by whole-cell voltage-clamp electrophysiology. The 3β-sulphates of pregnenolone (PREGS) and dehydroepiandrosterone (DHEAS) inhibited GlyR currents with K I values of 2–20 μM for different (α 1, α 2, α 4 and β) GlyR subunits. PREGS resulted in a parallel shift of the response curve of glycine for α 1 GlyRs. The inhibitory potencies of DHEAS relative to PREGS were decreased in transition from embryonic α 2 towards adult α 1β GlyRs. A decreased potency of DHEAS for α 4 versus α 2 GlyRs represents the first pharmacological difference reported between these subunits. A negative charge at C3 is required for GlyR antagonism but androsterone sulphate epimers at C3 inhibited without stereoselectivity. Some point mutations of α 1 GlyRs with characteristic functional consequences did not significantly affect the inhibitory potency of PREGS. Progesterone selectively inhibited α 2 GlyRs, while PREG and its acetic ester potentiated α 1 GlyRs. Coexpression of the α subunits with the β subunit eliminated the enhancing effects of PREG and attenuated the inhibitory potencies of the neurosteroids. Based on these data we propose that neurosteroids might modulate perinatal GlyR activity and thereby influence neuronal development.

Modulation of glycine receptors in retinal ganglion cells by zinc

Effects of zinc, an endogenous neuromodulator in the central nervous system, on glycine receptors (GlyRs) in retinal ganglion cells were investigated by using the whole-cell voltage-clamp technique. Zn2+ at low concentration (<2 microM) potentiated the glycine-induced chloride current and at higher concentration (>10 microM) suppressed it. This biphasic regulatory action of zinc acted selectively on the fast component of the glycine-induced current mediated by the strychnine-sensitive GlyRs, but not on the slow component mediated by the 5,7-dichlorokynurenic acid-sensitive GlyRs. Dose-response studies showed that 1 microM Zn2+ increased the maximum glycine response (I approximately) and shifted the EC50 to the left, suggesting that Zn2+ at low concentrations acts as an allosteric activator of the strychnine-sensitive GlyRs. Zn2+ at a concentration of 100 microM did not alter I approximately and shifted the EC50 to the right, indicating that Zn2+ at high concentrations acts as a competitive inhibitor of the GlyRs. Physiological functions of zinc modulation of GlyRs in retinal ganglion cells are discussed.

Prognostic clues and outcome of early treatment of nonketotic hyperglycinemia

Nonketotic hyperglycinemia presenting in the neonatal period is considered a devastating neurological condition. Most patients die soon after birth, and those who survive manifest severe neurological deficits, psychomotor retardation, and convulsions that are difficult to control. These abnormalities probably result from overstimulation of the N-methyl- d-aspartate receptor in the brain. In the present study, we wished to delineate prognostic clues for patients with neonatal (classic) nonketotic hyperglycinemia and to evaluate the results of early treatment with glycine-receptor modulators. The charts of six children treated in our department since 1991 were reviewed for details that might contribute to the prediction of prognosis. Postnatal clinical presentation and initial cerebrospinal fluid and plasma glycine levels were not predictive of outcome, but early treatment with N-methyl- d-aspartate modulators appeared to reduce late neurological complications and probably is as important as treatment with sodium benzoate. Attainment of developmental milestones, however, varies from one patient to another, presumably because of a factor or factors still unknown.