Glycine-evoked Currents Research Articles

Clinical, genetic, and functional characterization of the glycine receptor β-subunit A455P variant in a family affected by hyperekplexia syndrome

Hyperekplexia is a rare neurological disorder characterized by exaggerated startle responses affecting newborns with the hallmark characteristics of hypertonia, apnea, and noise or touch-induced nonepileptic seizures. The genetic causes of the disease can vary, and several associated genes and mutations have been reported to affect glycine receptors (GlyRs); however, the mechanistic links between GlyRs and hyperekplexia are not yet understood. Here, we describe a patient with hyperekplexia from a consanguineous family. Extensive genetic screening using exome sequencing coupled with autozygome analysis and iterative filtering supplemented by in silico prediction identified that the patient carries the homozygous missense mutation A455P in GLRB, which encodes the GlyR β-subunit. To unravel the physiological and molecular effects of A455P on GlyRs, we used electrophysiology in a heterologous system as well as immunocytochemistry, confocal microscopy, and cellular biochemistry. We found a reduction in glycine-evoked currents in N2A cells expressing the mutation compared to WT cells. Western blot analysis also revealed a reduced amount of GlyR β protein both in cell lysates and isolated membrane fractions. In line with the above observations, coimmunoprecipitation assays suggested that the GlyR α1-subunit retained coassembly with βA455P to form membrane-bound heteromeric receptors. Finally, structural modeling showed that the A455P mutation affected the interaction between the GlyR β-subunit transmembrane domain 4 and the other helices of the subunit. Taken together, our study identifies and validates a novel loss-of-function mutation in GlyRs whose pathogenicity is likely to cause hyperekplexia in the affected individual.

Ethanol consumption and sedation are altered in mice lacking the glycine receptor α2 subunit.

The precise mechanism/s of action of ethanol, although studied for many years, are not well understood. Like other drugs of abuse, ethanol affects dopamine levels in the nucleus accumbens (nAc), an important region of the mesolimbic system, causing a reinforcing effect. It has been shown that glycine receptors (GlyRs) present in the nAc are potentiated by clinically relevant concentrations of ethanol, where α1 and α2 are the predominant subunits expressed. Using a combination of electrophysiology and behavioural assays, we studied the involvement of GlyR α2 subunits on the effects of low and high doses of ethanol, as well as on consumption using mice lacking the GlyR α2 subunit (male Glra2-/Y and female Glra2-/- ). GlyR α2 subunits exist in accumbal neurons, since the glycine-evoked currents and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in Glra2-/Y mice were drastically decreased. In behavioural studies, differences in ethanol consumption and sedation were observed between wild-type (WT) and Glra2 knockout (KO) mice. Using the drinking in the dark (DID) paradigm, we found that Glra2-/Y mice presented a binge-like drinking behaviour immediately when exposed to ethanol rather than the gradual consumption seen in WT animals. Interestingly, the effect of knocking out Glra2 in female (Glra2-/- ) mice was less evident, since WT female mice already showed higher DID. The differences in ethanol consumption between WT and KO mice provide additional evidence supporting the conclusion that GlyRs are biologically relevant targets for the sedative and rewarding properties of ethanol.

Taurine activates glycine and GABAA receptor currents in anoxia-tolerant painted turtle pyramidal neurons.

Unlike anoxia-intolerant mammals, painted turtles can survive extended periods without oxygen. This is partly accomplished by an anoxia-mediated increase in gamma-aminobutyric acid (GABA) release, which activates GABA receptors and mediates spike arrest in turtle neurons via shunting inhibition. Extracellular taurine levels also increase during anoxia; why this occurs is unknown but it is speculated that glycine and/or GABAA/B receptors are involved. Given the general importance of inhibitory neurotransmission in the anoxia-tolerant painted turtle brain, we investigated the function of taurine as an inhibitory neuromodulator in turtle pyramidal neurons. Using whole-cell patch-clamp electrophysiological methods to record from neurons within a cortical brain sheet, we found that taurine depolarized membrane potential by ∼8 mV, increased whole-cell conductance ∼2-fold, and induced an inward current that possessed characteristics similar to GABA- and glycine-evoked currents. These effects were mitigated following glycine receptor antagonism with strychnine and GABAA receptor antagonism with gabazine, bicuculine or picrotoxin, but were unchanged following GABAB or glutamatergic receptor inhibition. These data indicate that a high concentration of taurine in vitro mediates its effects through both glycine and GABAA receptors, and suggests that taurine, in addition to GABA, inhibits neuronal activity during anoxia in the turtle cortex.

Presence of ethanol-sensitive glycine receptors in medium spiny neurons in the mouse nucleus accumbens.

The nucleus accumbens (nAc) is involved in addiction-related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse. Alcohol abuse causes major social, economic and health-related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and β in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)-positive MSNs. Interestingly, the glycine-evoked currents in dissociated DRD1-positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR-mediated tonic current by ethanol suggests that they modulate the excitability of DRD1-positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction-related and compulsive behaviours.

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the α2 or α3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala(254)) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala(254) determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level.

Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn2+, together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition – a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.

ON-pathway-dominant glycinergic regulation of cholinergic amacrine cells in the mouse retina.

Direction selectivity in the retina has been studied as a model of dendritic computation of neural circuits. Starburst amacrine cells (SACs) have been examined as a model system of dendritic computation as they play a pivotal role in the formation of direction selectivity. Because the difference of anatomical location inside the retina made ON-SACs an easier target to record, the biophysical properties of ON-SACs have been used to predict those of OFF-SACs. In this study, we systematically compared the responses of ON- and OFF-SACs to the two principal neurotransmitters, glycine and glutamate. We found that responses to glycine were significantly larger in ON-SACs than in OFF-SACs. In contrast, ON- and OFF-SACs responded similarly to glutamate. The amplitude of glycine responses in ON-SACs increased after eye opening and the largest amplitude was observed at postnatal day 28. On the other hand, no increase in the amplitude of glycine responses in OFF-SACs was observed until postnatal day 28. Glycine-evoked currents were inhibited by the application of strychnine. Glutamate-evoked currents were mimicked by the application of AMPA or kainite, and responses to N-methyl-d-aspartate were observed in the absence of Mg(2+) block. Glutamate-evoked currents produced an increase in the frequency of GABAergic inhibitory postsynaptic currents. Our results suggest that signal processing in ON-SACs cannot be directly used to understand the properties of OFF-SACs. Therefore fully defining the physiological properties of OFF-SACs will be critical to understanding and modelling direction selectivity in the retina.

The Activation Mechanism of Rat α3 Homomeric Glycine Receptors

The α3-containing glycine channels (GlyR) are found in discrete areas of the spinal cord and hippocampus, but despite their likely physiological relevance, their kinetic properties are unknown. We investigated the activation mechanism of recombinant α3 rat homomeric glycine receptors. Cell-attached steady-state single channel recordings were obtained at 50 - 10000 μM glycine. Macroscopic synaptic-like glycine-evoked currents were obtained by applications of pulses of glycine (1 ms, 10 mM) to outside-out patches (intracellular chloride concentration 20 mM). Kinetic mechanisms were tested using maximum likelihood fits by the HJCFIT program to sets of idealized single channel records. The adequacy of each mechanism was judged by comparing the predictions of the model with the summary statistics of the single channel data and the time course of macroscopic deactivation.The single channel open probability of homomeric α3 GlyR was strongly concentration-dependent, with a Hill slope of of 3.7 ± 0.1, much steeper than that of α GlyRs (1.82 ± 0.24, Beato et al., 2004). This suggests that α3 GlyR require all five binding sites to bind glycine in order to reach their maximum open probability. In other homomeric Cys-loop channels, including α GlyR, occupancy of three out of five sites is sufficient.Other features of a3 GlyR activation were similar to those of other GlyR. In particular, the fully-liganded opening rate constant was 150,000 ± 24,000 s-1 and the overall efficacy was 67 ± 4. The microscopic affinity of glycine for the intermediate shut “flip” conformation was 160 ± 24 μM, approximately 5-fold higher than for the resting conformation (890 ± 80 μM; n = 3 sets). This accounted for the apparent cooperativity of the response.Beato, Groot-Kormelink, Colquhoun & Sivilotti (2004). J Neurosci 24, 895.

Mutations M287L and Q266I in the Glycine Receptor α1 Subunit Change Sensitivity to Volatile Anesthetics in Oocytes and Neurons, but Not the Minimal Alveolar Concentration in Knockin Mice

Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. We produced single amino acid mutations into the glycine receptor α1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brainstem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. Studies of glycine-evoked currents in brainstem neurons from knockin mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.

Interaction between taurine and GABAA/glycine receptors in neurons of the rat anteroventral cochlear nucleus

Taurine, one of the most abundant endogenous amino acids in the mammalian central nervous system (CNS), is involved in neural development and many physiological functions. In this study, the interaction between taurine and GABAA/glycine receptors was investigated in young rat (P13–P15) anteroventral cochlear nucleus (AVCN) neurons using the whole-cell patch-clamp method. We found that taurine at low (0.1mM) and high (1mM) concentrations activated both GABAA and glycine receptors, but not AMPA and NMDA receptors. The reversal potentials of taurine-, GABA- or glycine-evoked currents were close to the expected chloride equilibrium potential, indicating that receptors activated by these agonists were mediating chloride conductance. Moreover, our results showed that the currents activated by co-application of GABA and glycine were cross-inhibitive. Sequential application of GABA and glycine or vice versa also reduced the glycine or GABA evoked currents. There was no cross-inhibition when taurine and GABA or taurine and glycine were applied simultaneously, but the response was larger than that evoked by GABA or glycine alone. These results suggest that taurine can serve as a neuromodulator to strengthen GABAergic and glycinergic neurotransmission in the rat AVCN.

Glycine Receptor Channels in Spinal Motoneurons Are Abnormal in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). In this study, we have examined, using whole-cell patch-clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing enhanced green fluorescent protein (eGFP) driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP-expressing (Hb9-eGFP(+)) neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP(+) motoneurons prepared from G93A-SOD1 embryos and from their nontransgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature IPSCs (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell reverse transcription-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.

A Salt Bridge Crucial for Glycine Receptor Function

The glycine receptor (GlyR) chloride channel is a member of the pentameric ligand-gated ion channel (pLGIC) receptor family. These channels mediate fast inhibitory transmission in the nervous system. Recent structural data and a wide body of functional data have underlined the importance of the interface between the ligand-binding domain and the transmembrane domain in channel gating. A number of charged residues at this interface have been shown to form salt bridges critical for channel function in other pLGICs. However, no such interaction has been demonstrated in the GlyR. Mutations at D148 (Cys-loop) and R218 (pre-M1) in the homomeric alpha 1 GlyR can have drastic effects on GlyR function and the single mutants D148C and R218C failed to produce glycine-induced currents in Xenopus oocytes. However, the D148C, R218C double mutant resulted in receptors that produced glycine-gated currents with only a small decrease in glycine sensitivity compared to WT. Importantly, sub-saturating glycine-evoked currents were dramatically increased by the reducing agent dithiothreitol. This suggests that D148C and R218C can form a disulphide bridge, possibly indicating an electrostatic interaction between D148 and R218 that stabilizes the channel closed state. The possibilty of a state-dependent salt bridge between D148 and R218 raised the question how the charges are stabilised in the open state. Interestingly, structural studies have suggested that R218 is flanked by two highly conserved phenylalanine side chains. To test, if these aromatic amino acids interact with R218 electrostically via a cation-pi interaction, we incorporated a series of fluorinated Phe derivatives using the in vivo non-sense suppression method. However, the fluorination-induced changes in the glycine sensitivity were not consistent with a cation-pi interaction. Taken together, these results point towards a crucial salt bridge between D148 and R218 but no electrostatic contributions by the flanking phenylalanines.

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

Contrary to its effect on the gamma-aminobutyric acid type A and C receptors, picrotoxin antagonism of the alpha1 homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and nonselective between the picrotoxin components picrotoxinin and picrotin. Picrotoxin antagonism of the embryonic alpha2 homomeric GlyR is known to be use-dependent and reflects a channel-blocking mechanism, but the selectivity of picrotoxin antagonism of the embryonic alpha2 homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin- and picrotoxinin-induced inhibition of currents, which were evoked by the activation of alpha2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents, picrotin had a 30 times higher IC50 than picrotoxinin. Picrotin-evoked inhibition displayed voltage dependence, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentration-dependent manner, indicating that these picrotoxin components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were co-applied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but both could be trapped at their binding site when the channel closed during glycine dissociation. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking alpha2 homomeric GlyR.

Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine-proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.

Mechanisms for Picrotoxin Block of α2 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.

Serotonin, l‐tryptophan, and tryptamine are effective inhibitors of the amino acid transport system PAT1

The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of l- and d-proline, l-alanine, glycine, taurine, d-serine, GABA, and many other related compounds and drugs. Here we describe the novel finding that l-tryptophan and its derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H+-dependent l-[3H]proline uptake into Caco-2 cells with inhibition constants (K(i)) of 0.9 to 6.1 mM. Uptake of l-[3H]tryptophan into Caco-2 cells on the other hand was not inhibited by l-proline. Whereas PAT1 substrates produced significant changes in a membrane potential assay for electrogenic transport in Caco-2 cells, l-tryptophan, tryptamine, and 5-hydroxy-l-tryptophan failed to alter membrane voltage. When PAT1 was expressed in Xenopus laevis oocytes and analyzed by the two-electrode voltage clamp technique, glycine elicited high inward currents that were dependent on membrane potential but no currents were observed with l-tryptophan, tryptamine, 5-hydroxy-l-tryptophan, or serotonin. Although not transported electrogenically by PAT1, l-tryptophan and its derivatives inhibited glycine-evoked currents dose-dependently. We conclude that serotonin, l-tryptophan, and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers' naturally occurring inhibitors that may alter the transport function of PAT1.

A novel physiological mechanism of glycine-induced immunomodulation: Na+-coupled amino acid transporter currents in cultured brain macrophages.

Glycine is known to modulate immune cell responses. However, the physiological mechanisms underlying inhibitory effects of glycine on macrophages are not well understood. Here we show that glycine is capable of inducing inward currents in brain macrophages (microglia). In contrast to glycine, the glycine receptor agonist taurine failed to elicit currents. Glycine-evoked currents of brain macrophages were unaffected by strychnine, Cl(-)-free extracellular solution, N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (NFPS) and amoxapine, but were abolished upon omission of extracellular Na(+). Furthermore, glycine caused increases in the intracellular Na(+) concentration and pronounced membrane depolarization. Glycine-evoked depolarization was Na(+) dependent and occurred independently of the intracellular Cl(-) concentration. Similarly to glycine, glutamine and alpha-(methylamino)isobutyric acid (MeAIB) elicited inward currents in brain macrophages. In the presence of either glutamine or MeAIB, glycine-induced currents were inhibited. It is concluded that neither functional glycine receptors nor glycine transporters are expressed in brain macrophages. We suggest that glycine mediates its effects by activation of system A Na(+)-coupled neutral amino acid transporters.

Mechanisms of H+ modulation of glycinergic response in rat sacral dorsal commissural neurons.

Many ionotropic receptors are modulated by extracellular H+. So far, few studies have directly addressed the role of such modulation at synapses. In the present study, we investigated the effects of changes in extracellular pH on glycinergic miniature inhibitory postsynaptic currents (mIPSCs) as well as glycine-evoked currents (IGly) in mechanically dissociated spinal neurons with native synaptic boutons preserved. H+ modulated both the mIPSCs and IGly biphasically, although it activated an amiloride-sensitive inward current by itself. Decreasing extracellular pH reversibly inhibited the amplitude of the mIPSCs and IGly, while increasing external pH reversibly potentiated these parameters. Blockade of acid-sensing ion channels (ASICs) with amiloride, the selective antagonist of ASICs, or decreasing intracellular pH did not alter the modulatory effect of H+ on either mIPSCs or IGly. H+ shifted the EC50 of the glycine concentration-response curve from 49.3 +/- 5.7 microM at external pH 7.4 to 131.5 +/- 8.1 microM at pH 5.5, without altering the Cl- selectivity of the glycine receptor (GlyR), the Hill coefficient and the maximal IGly, suggesting a competitive inhibition of IGly by H+. Both Zn2+ and H+ inhibited IGly. However, H+ induced no further inhibition of IGly in the presence of a saturating concentration of Zn2+. In addition, H+ significantly affected the kinetics of glycinergic mIPSCs and IGly. It is proposed that H+ and/or Zn2+ compete with glycine binding and inhibit the amplitude of glycinergic mIPSCs and IGly. Moreover, binding of H+ induces a global conformational change in GlyR, which closes the GlyR Cl- channel and results in the acceleration of the seeming desensitization of IGly as well as speeding up the decay time constant of glycinergic mIPSCs. However, the deprotonation rate is faster than the unbinding rate of glycine from the GlyR, leading to reactivation of the undesensitized GlyR after washout of agonist and the appearance of a rebound IGly. H+ also modulated the glycine cotransmitter, GABA-activated current (IGABA). Taken together, the results support a "conformational coupling" model for H+ modulation of the GlyR and suggest that H+ may act as a novel modulator for inhibitory neurotransmission in the mammalian spinal cord.

Decay of ethanol-induced suppression of glycine-activated current of ventral tegmental area neurons

We demonstrated previously that ethanol depresses glycine-induced currents in 45% of neurons freshly isolated from the ventral tegmental area (VTA) of rats ( Ye et al., 2001b), and that protein kinase C (PKC) modulates this action of ethanol ( Tao and Ye, 2002). In the present study, we investigated the time course of this effect of ethanol on VTA neurons from young rats. For 70% of the neurons in which ethanol reduced glycine-evoked currents, this depressant effect gradually diminished during continuous superfusion with ethanol. Its action decayed faster when ethanol was applied in several brief pulses than by continuous superfusion. On the other hand, the decay was especially slower when ethanol was applied in pulses at longer intervals or by preincubation. Phorbol ester 12,13-dibutyrate (PDBu, 1 μM), an activator of PKC, also depressed glycine-induced currents. In ~40% (6/15) of the neurons, the effect of PDBu diminished with time and was antagonized by the specific PKC inhibitor, chelerythrine (7μM). Chelerythrine also attenuated the ethanol-induced depression of glycine-induced currents and its time-dependent decay, thus confirming our previous evidence that PKC mediates, at least in part, the decay of the depressant effect of ethanol on glycine-induced currents of VTA neurons.