A minimal model to account for the response of N-Methyl-D-aspartate receptors expressed in Xenopus oocyte injected with rat brain mRNA.

N-Methyl-D-aspartate (NMDA) receptors were expressed in Xenopus oocytes by injecting rat brain mRNA. NMDA-elicited responses in the oocytes were measured by the voltage-clamping method. The following measurements were made in the presence of 50 microM glycine (Gly) to establish the relationship between the NMDA concentration and the current: (1) the NMDA-induced membrane current before desensitization; (2) the NMDA-induced membrane current after desensitization equilibrium; (3) the fraction of the active form of the receptor after desensitization equilibrium in the presence and absence of 50 microM Gly; (4) the rate of the recovery of desensitized receptors upon removal of NMDA. Gly was essential for not only the activation of NMDA receptors but also their desensitization. These results were analyzed on the basis of a minimal model where one agonist and one Gly binding site were assumed. The equilibrium and rate constants of the model were evaluated for NMDA in the presence of saturating amounts of Gly. This model will be useful for systematically explaining the complicated responses of NMDA receptors.     

Acetylcholine receptor-mediated membrane current in oocytes injected with Electrophorus electricus mRNA: analyses of nicotine, succinylcholine, and decamethonium responses on the basis of the minimal model

Nicotinic acetylcholine receptor was synthesized in Xenopus oocytes after injection of the mRNA purified from Electrophorus electricus electroplax. Nicotine, succinylcholine, and decamethonium (agonist)-elicited membrane currents in the injected oocytes were measured electrophysiologically by the voltage-clamping method. The following four different measurements were made to establish the relationship between the agonist concentration and the membrane current: 1) the agonist-induced membrane current before desensitization, 2) the agonist-induced membrane current after desensitization equilibrium, 3) the fraction of the active form of the receptors after desensitization equilibrium, 4) the rate of recovery of desensitized receptors upon removal of the agonist. These results were analyzed on the basis of the minimal model proposed from receptor-mediated ion translocation measurements. The equilibrium and rate constants of the model were evaluated for nicotine, succinylcholine, and decamethonium, and could explain the observed electrical responses in the injected oocyte, i.e. the characteristics of the receptor response caused by these agonists.     

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.     

The general anesthetic pentobarbital slows desensitization and deactivation of the glycine receptor in the rat spinal dorsal horn neurons

Although many general anesthetics have been found to produce anesthetic and analgesic effects by augmenting GABA(A) receptor (GABA(A)R) function, the role of the glycine receptor (GlyR) in this process is not fully understood at the neuronal level in the spinal cord. We investigated the effects of a barbiturate general anesthetic, pentobarbital (PB), on the glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and the responses to exogenously applied glycine, or taurine, a low affinity GlyR agonist, by using the whole-cell patch-clamp technique in the rat spinal dorsal horn neurons isolated using a novel mechanical method. Bath application of 30 microm PB significantly prolonged the decay time constant of the spontaneous glycinergic mIPSC without changing its amplitude and frequency. Co-application of 0.3 mm PB reduced the peak amplitude, affected the macroscopic desensitization and deactivation of the response to externally applied Gly in a concentration-dependent manner. In addition, the recovery of Gly response from desensitization was also prolonged by PB. However, PB did not change the desensitization and deactivation kinetics of the taurine-induced response. The GABA(A)R antagonist bicuculline (10 microm) did not affect the effect of PB on the Gly response. Thus, PB prolonged the spinal glycinergic mIPSCs by slowing desensitization and deactivation of GlyR. Two other structurally different intravenous anesthetics, i.e. propofol (10 microm) and etomidate (3 microm), prolonged the duration of the glycinergic mIPSC in the rat spinal dorsal horn neurons. In conclusion, on GlyR-Cl(-) channel complexes there may exist action site(s) of intravenous general anesthetics. GlyR and glycinergic neurotransmission may play an important role in the modulation of general anesthesia in the mammalian spinal cord.     

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

Noncompetitive inhibition of acetylcholine receptor-controlled ion translocation was studied in membrane vesicles prepared from both Torpedo californica and Electrophorus electricus electroplax. Ion flux was measured in the millisecond time region by using a spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, and a quench-flow technique using 86Rb+. The rate coefficient of ion flux prior to receptor inactivation (desensitization), JA, was measured at different acetylcholine and inhibitor concentrations, in order to assess which active (nondesensitized) receptor forms bind noncompetitive inhibitors. The degree of inhibition of JA by the inhibitors studied (cocaine, procaine, and phencyclidine) was found to be independent of acetylcholine concentration. The results are consistent with a mechanism in which each compound inhibits by binding to a single site that exists with equal affinity on all active receptor forms. Mechanisms in which the inhibitors bind exclusively to the open-channel form of the receptor are excluded by the data. The same conclusions were reached in cocaine experiments at 0-mV and procaine experiments at -25-mV transmembrane voltage in T. californica vesicles. It had been previously shown that phencyclidine, in addition to decreasing JA (by binding to active receptors), also increases the rate of rapid receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptors (by binding better to inactivated receptor than to active receptor in the closed or open conformations). These effects were not observed with cocaine or procaine. Here it is shown that despite these differential effects on inactivation, cocaine and phencyclidine bind to the same inhibitory site on active receptors (in E. electricus vesicles).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA

The effect of 13-L-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li+ and 45Ca2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and gamma-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.     

Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C

Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor-gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR-gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity.     

Potentiation and Inhibition of Ionotropic Neurotransmitter Receptors Expressed in Xenopus Oocyte by Linoleic Acid and Its Hydroperoxide

Abstract: To study the effects of lipid hydroperoxide on ionotropic neurotransmitter receptors, γ-aminobutyric acid (GABA), N -methyl- d -aspartate (NMDA), and non-NMDA receptors (GABARs, NMDARs, and non-NMDARs, respectively) were expressed in Xenopus oocytes that received an injection of mRNA prepared from rat whole brain. Linoleic acid (LA) and its hydroperoxide 13- l -hydroperoxylinoleic acid (LOOH) prepared with soybean lipoxygenase inhibited the response of GABARs in the presence of GABA at high concentrations. The inhibition was stronger when the inhibitors were perfused 1 min before a mixture of GABA and the inhibitors than when they were perfused simultaneously with GABA. On the other hand, only LOOH potentiated the response of GABARs in the presence of GABA at low concentrations, possibly increasing the affinity of GABA to the receptors. Both LA and LOOH accelerated the rate of desensitization of GABARs, but LOOH did not affect their equilibrium between the active and desensitized form of the receptors. They also inhibited the response of NMDARs in a noncompetitive manner but barely inhibited the response of non-NMDARs in the presence of kainate at various concentrations. These results suggest the possibility that production of lipid hydroperoxide modulates the neural transmission in the brain, especially through GABARs.     

Minimal model to account for the membrane conductance increase and desensitization of gamma-aminobutyric acid receptors synthesized in the Xenopus oocytes injected with rat brain mRNA

gamma-Aminobutyric acid (GABA) receptors, which translocate chloride anion with binding GABA, were synthesized in Xenopus oocytes by injecting rat brain mRNA. GABA-elicited responses in the oocytes were measured electrophysiologically by the current-clamped method. Five different measurements were made to establish the relationship between GABA concentration and the electrical responses: (1) the GABA-elicited conductance increase before desensitization; (2) the rate of desensitization of GABA receptors; (3) the rate of recovery of desensitized receptors upon removal of GABA; (4) the GABA-elicited conductance increase after desensitization equilibrium; (5) the fraction of the active form of GABA receptors after desensitization equilibrium. These results were interpreted on the basis of the minimal model proposed for nicotinic acetylcholine receptor in Electrophorus electricus electroplax [Hess, G. P., Cash, D. J., & Aoshima, H. (1983) Annu. Rev. Biophys. Bioeng. 12, 443-473]. Estimated equilibrium and rate constants in the model for GABA receptors could successfully explain the results of the five above measurements.     

Characteristics of chloride-dependent effects of acetylcholine and neutral aminoacids on neurons of the snail (Achatina)

The response was investigated of neurons composing the cerebral ganglia inAchatina fulica (the Giant African snail) to application of acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly). Chloride-dependent currents induced by these transmitters in 1 1/2-month old siblings were inhibited by dibutyryl-cAMP and strychnine. Inhibition of ACh response produced 10^–8 M GABA was mimicked by application of dibutyryl-cAMP and isobutylmethylxanthine. Complete cross-desensitization was characteristic of both GABA- and Bly-induced response, but this effect did not occur when ACh and GABA (or Gly) were applied. A conclusion was reached on the basis of the pharmacological relationship between GABA- and Gly-induced response that these amino acids act on a single receptor — channel complex in the neurons of infant snails, whereas ACh-, GABA-, and Gly-induced chloride currents were not so related in cells of 4 year-oldAchatina.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 35–43, January–February, 1991.     

The influence of glutamic and aminoacetic acids on the excitability of the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that a resting potential (RP), an action potential (AP) and a calcium component of AP (named voltage transient, VT) can be influenced by glutamic acid (Glu) and aminoacetic acid (glycine, Gly) in the liverwort Conocephalum conicum. In the continuous presence of 5mM Glu or 5mM Gly, the RP hyperpolarized constantly and the plants became desensitized to the excitatory amino acids (Glu or Gly). Under such circumstances, the amplitudes of APs evoked by stimuli other than Glu or Gly grew, as did their calcium components (VTs). The sudden application of 1-15 mM Glu or Gly to a thallus not yet desensitized resulted in an excitation, i.e. a single AP or AP series. Aspartate (Asp) could not substitute for Glu in any way. Simultaneous action of both amino acids acted synergically to trigger APs. The same phenomenon was observed when glycine solution was enriched with N-methyl-D-aspartic acid (NMDA). Gly-induced APs were totally hindered by 1mM D-amino-5-phosphonopentanoic acid (AP5)--an inhibitor of ionotropic glutamate receptors of the NMDA kind. Glu-induced APs could be totally suppressed by 1mM AP5 as well as by 1mM 6,7-dinitroquinoxaline-2,3-dione (DNQX)--an inhibitor of AMPA/KA receptors. DNQX also completely blocked the calcium component of Glu-evoked APs. After DNQX treatment, the only response to Glu was a membrane potential hyperpolarization (like the Glu response in a desensitized plant). It was concluded that the Glu-induced depolarization and hyperpolarization are separate phenomena. The stimulatory effects of both Glu and Gly on liverwort excitability may be the consequences of an activation of a variety of ionotropic Glu receptor subtypes.     

The mammalian glycine receptor: biology and structure of a neuronal chloride channel protein

Glycine is a major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates and invertebrates. The postsynaptic receptor for this amino acid is an oligomeric glycoprotein which, upon binding of glycine, transiently forms an anion-selective transmembrane channel. Agonist-mediated receptor activation is antagonized by strychnine, a high-affinity ligand of the glycine receptor (GlyR). Biochemical and immunological data show that affinity-purified preparations of the mammalian GlyR contain three polypeptides of M_r 48,000, 58,000 and 93,000. These polypeptides have different functional properties and/or topologies in the postsynaptic membrane of the glycinergic synapse. The primary sequence of the M_r 48,000 subunit deduced by cDNA cloning exhibits structural and amino-acid homology to nicotinic acetylcholine and GABA_a receptor proteins, indicating a common evolutionary relationship between the different neurotransmitter-gated ion channels of excitable membranes. Monoclonal antibodies against the GlyR allow its histochemical localization in different regions of the CNS. GlyR deficiencies have been implicated in the pathogenesis of spasticity and spinal cord degeneration in mouse and man.     

The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system

The postsynaptic glycine receptor (GlyR) is a major inhibitory chloride channel protein in the central nervous system. The affinity-purified receptor contains polypeptides of 48 kDa, 58 kDa, and 93 kDa. The 48-kDa (alpha) and 58 kDa (beta) subunits span the postsynaptic membrane in a pentameric arrangement to form the anion channel of the receptor. The 93-kDa polypeptide is cytoplasmically localized and may have an anchoring function. Molecular cloning revealed that different structural characteristics are shared by the membrane-spanning subunits of the GlyR and those of other ligand-gated ion channel proteins. Developmental regulation of the GlyR is characterized by alterations in antagonist binding, heterogeneity of alpha subunits, and increased levels of the 93-kDa polypeptide. Glycine receptor function can be reconstituted by expression of cloned alpha subunits in heterologous cell systems. Positive charges found at the presumed mouths of the GlyR channel appear to be important determinants of ion selectivity. These data establish the anion-conducting GlyR as a homolog of other ligand-gated ion channel proteins and suggest that the diversity of these channels originates from divergent evolution of a primordial channel protein early in phylogeny.     

Cytosolic acidification and intracellular zinc release in hippocampal neurons

In neurons exposed to glutamate, Ca2? influx triggers intracellular Zn2? release via an as yet unclear mechanism. As glutamate induces a Ca2?-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca2? concentration ([Ca2?](i)), intracellular pH (pH(i) ), and [Zn2?](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn2?](i), [Ca2?](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn2? release in a Ca2?-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn2?. The rate of Glu/Gly-induced [Zn2?](i) increase was not correlated with the rate of [Ca2?](i) increase. Instead, the extent of [Zn2?](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn2?](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca2?-dependent pH(i) drop (plasmalemmal Ca2? pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn2? release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn2? release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca2? influx or glutamate receptor activation) led to intracellular Zn2? release; the released Zn2? (free Zn2? plus Zn2?) bound to Fura-2FF and FluoZin-3) reached 1 μM.     

Molecular biology of glycinergic neurotransmission

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem of vertebrates. Glycine is accumulated into synaptic vesicles by a proton-coupled transport system and released to the synaptic cleft after depolarization of the presynaptic terminal. The inhibitory action of glycine is mediated by pentameric glycine receptors (GlyR) that belong to the ligand-gated ion channel superfamily. The synaptic action of glycine is terminated by two sodium- and chloride-coupled transporters, GLYT1 and GLYT2, located in the glial plasma membrane and in the presynaptic terminals, respectively. Dysfunction of inhibitory glycinergic neurotransmission is associated with several forms of inherited mammalian myoclonus. In addition, glycine could participate in excitatory neurotransmission by modulating the activity of the NMDA subtype of glutamate receptor. In this article, we discuss recent progress in our understanding of the molecular mechanisms that underlie the physiology and pathology of glycinergic neurotransmission.     

Mechanisms for picrotoxin block of alpha2 homomeric glycine receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.     

NH(2)-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

A synthetic, channel-forming peptide, derived from the alpha-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH(2) terminus (N-K(4)-M2GlyR). In Ussing chamber experiments, apical N-K(4)-M2GlyR (250 microM) increased transepithelial short-circuit current (I(sc)) by 7.7 +/- 1.7 and 10.6 +/- 0.9 microA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K(4)-M2GlyR caused a concentration-dependent increase in I(sc) (k([1/2]) = 190 microM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K(4)-M2GlyR-mediated increases in I(sc) were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl(-) conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K(4)-M2GlyR-mediated I(sc). Whole cell membrane patch voltage-clamp studies revealed an N-K(4)-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K(4)-M2GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl(-) of 290 meq. These results indicate that N-K(4)-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.     

Kinetic analysis of recombinant mammalian α<Subscript>1</Subscript> and α<Subscript>1</Subscript>β glycine receptor channels

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.     

Glycine attenuates cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis in mice

Glycine is a cytoprotector to protect cells against ischemic damage by counteracting neuronal depolarization. However, whether it can directly inhibit neuronal apoptosis is unknown. In this study, we demonstrated that glycine could attenuate ischemia/reperfusion (I/R) induced cerebral infarction and improved neurological outcomes in mice. The protective effect of glycine was associated with reduction of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) positive cells, deactivation of phosphor-JNK, inhibition of caspase-3 cleavage, down-regulation of FasL/Fas, and up-regulation of bcl-2 and bcl-2/bax in the mouse I/R penumbra. The beneficial effect of glycine against oxygen and glucose deprivation (OGD) induced injury was also confirmed in SH-SY5Y cells as well as in primary cultured neurons, which was significantly dampened by knockdown of glycine receptor α1 (GlyR α1) with siRNA transfection or by preventing glycine binding with glycine receptor using a specific antibody against glycine receptor. These results suggest that glycine antagonize cerebral I/R induced injury by inhibiting apoptosis in mice. Glycine could block both extrinsic and intrinsic apoptotic pathways for which GlyR may be required.     

Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor.

The inhibitory postsynaptic glycine receptor (GlyR) of rat spinal cord is an oligomeric transmembrane protein which forms an agonist-gated anion channel. Expression in Xenopus oocytes of its mol. wt 48,000 subunit generated glycine-gated chloride channels which were analysed by voltage clamp. The agonist and antagonist response properties as well as the desensitization characteristics of these 48 kd subunit receptors resembled GlyRs expressed from spinal cord poly(A)+ RNA. These data indicate that the 48 kd subunit is capable of assembling into a functional receptor homo-oligomer which displays the pharmacology characteristic of the spinal cord GlyR.