Modulation of glycine receptor single-channel conductance by intracellular phosphorylation.

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.

Large Intracellular Domain-Dependent Effects of Positive Allosteric Modulators on Glycine Receptors.

Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α1GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.

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

KEY POINTS: 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. ABSTRACT: 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 ß 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.

[The post-synaptic glycine receptor mediates in a transitory and sustained way the glycinergic inhibition in the vertebrate retina]. / El receptor postsinaptico de glicina media de forma transitoria y sostenida la inhibicion glicinergica en la retina de los vertebrados.

INTRODUCTION: Glycine and the gamma-aminobutyric acid are the principal inhibitory neurotransmitters in the vertebrate retina. The inhibitory action of glycine is mediated by the post-synaptic glycine receptor, a chloride-selective channel, constituted by three beta and two alpha subunits (alpha(1)-alpha(4)), which is antagonized by the alkaloid strychnine. In the retina, it is known that all alpha isoforms are expressed at the level of the inner synaptic layer with a very low colocalization. The glycine receptor formed by either alpha1 or alpha(3) shows rapid kinetics, whereas alpha(2) or alpha(4) receptors respond tonically. The use of transgenic mice has allowed the study of the different glycine receptor alpha subunits in the glycinegic neurotransmission of the mammalian retina. AIM: To describe the participation of the glycine receptor in the inhibitory neurotransmission particularly in the retina. DEVELOPMENT: In this review we describe the experiments that have allowed the localization and the involvement of the alpha subunit isoforms in specific transmission circuits of the vertebrate retina. CONCLUSIONS: The localization of the glycine receptor conformed by different isoforms of the alpha subunit in specific neuronal types, indicate the presence of glycinergic circuits that encode information differently in the retina.

Anti-homeostatic synaptic plasticity of glycine receptor function after chronic strychnine in developing cultured mouse spinal neurons.

In this study, we describe a novel form of anti-homeostatic plasticity produced after culturing spinal neurons with strychnine, but not bicuculline or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Strychnine caused a large increase in network excitability, detected as spontaneous synaptic currents and calcium transients. The calcium transients were associated with action potential firing and activation of gamma-aminobutyric acid (GABA(A)) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as they were blocked by tetrodotoxin (TTX), bicuculline, and CNQX. After chronic blockade of glycine receptors (GlyRs), the frequency of synaptic transmission showed a significant enhancement demonstrating the phenomenon of anti-homeostatic plasticity. Spontaneous inhibitory glycinergic currents in treated cells showed a fourfold increase in frequency (from 0.55 to 2.4 Hz) and a 184% increase in average peak amplitude compared with control. Furthermore, the augmentation in excitability accelerated the decay time constant of miniature inhibitory post-synaptic currents. Strychnine caused an increase in GlyR current density, without changes in the apparent affinity. These findings support the idea of a post-synaptic action that partly explains the increase in synaptic transmission. This phenomenon of synaptic plasticity was blocked by TTX, an antibody against brain-derived neurotrophic factor (BDNF) and K252a suggesting the involvement of the neuronal activity-dependent BDNF-TrkB signaling pathway. These results show that the properties of GlyRs are regulated by the degree of neuronal activity in the developing network.

Molecular determinants for G protein betagamma 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.

Glycinergic and GABAergic synaptic transmission are differentially affected by gephyrin in spinal neurons.

In the present study, we have examined the physiological properties of synaptic currents mediated by GlyRs and GABAARs after culturing spinal neurons with a gephyrin antisense oligonucleotide. Application of gephyrin antisense, but not the sense, reduced the glycinergic mIPSC amplitude ( approximately 50%) and frequency ( approximately 85%), indicating the importance of gephyrin for GlyR anchoring at postsynaptic sites. On the other hand, the glycine-evoked current amplitude was unchanged indicating that functional GlyRs were still located in the extrasynaptic membrane. The analysis of the GABAergic transmission in the same neurons revealed approximately 70% reduction in the frequency of the GABAergic mIPSCs, without changes in the amplitude. Interestingly, the modulation of remaining GABAAR-mediated synaptic events by zinc and diazepam was significantly altered by the antisense. These results indicate that gephyrin is required for the membrane insertion/stabilization of the GABAAR gamma2 subunit as well as for its subsequent localization in the postsynaptic membrane.

Developmental-dependent action of microtubule depolymerization on the function and structure of synaptic glycine receptor clusters in spinal neurons.

Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. However, the consequence of microtubule disruption on the structure of postsynaptic GlyR/gephyrin clusters is controversial and possible alterations in function are largely unknown. In this study, we have examined the physiological and morphological properties of GlyR/gephyrin clusters after colchicine treatment in cultured spinal neurons during development. In immature neurons (5-7 DIV), disruption of microtubules resulted in a 33 +/- 4% decrease in the peak amplitude and a 72 +/- 15% reduction in the frequency of spontaneous glycinergic miniature postsynaptic currents (mIPSCs) recorded in whole cell mode. However, similar colchicine treatments resulted in smaller effects on 10-12 DIV neurons and no effect on mature neurons (15-17 DIV). The decrease in glycinergic mIPSC amplitude and frequency reflects postsynaptic actions of colchicine, since postsynaptic stabilization of microtubules with GTP prevented both actions and similar reductions in mIPSC frequency were obtained by modifying the Cl(-) driving force to obtain parallel reductions in mIPSC amplitude. Confocal microscopy revealed that colchicine reduced the average length and immunofluorescence intensity of synaptic gephyrin/GlyR clusters in immature (approximately 30%) and intermediate (approximately 15%) neurons, but not in mature clusters. Thus the structural and functional changes of postsynaptic gephyrin/GlyR clusters after colchicine treatment were tightly correlated. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both alpha2beta and alpha1beta GlyRs in immature neurons to only alpha1beta GlyRs in mature neurons. Microtubule disruption preferentially affected postsynaptic sites containing alpha2beta-containing synaptic receptors.

Modulation of glycine-activated ion channel function by G-protein betagamma subunits.

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.

Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca(2+) at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABA(A) and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 microM), nimodipine (4 microM) and bicuculline (20 microM) completely blocked it. On the other hand, APV (50 microM) and CNQX (20 microM) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

Modulation of defensive behavior by periaqueductal gray NMDA/glycine-B receptor.

Glutamate (GLU) associated with glycine, act as co-transmitter at the N-methyl-D-aspartate/glycine-B (NMDA/GLY(B)) receptor. Dorsal periaqueductal gray (dPAG) neurons express NMDA/GLY(B) receptors suggesting a GLU physiological role in mediating the responses elicited by stimulation of this area. Immunohistochemical data provided evidence of a possible correlation among elevated plus-maze (EPM), fear-like defensive behavior, and dPAG activity. The present data show that whereas the NMDA/GLY(B) receptor agonists increased the open-arm avoidance responses in the EPM, the antagonists had the opposite effects. Microinjection of NMDA/GLY(B) receptor agonists within the dPAG during test sessions in the EPM resulted in an enduring learned fear response detected in the retest. Therefore, in addition to the proposed role for the dPAG in panic attacks (escape), these findings suggest that the dPAG can also participate in more subtle anxiety-like behaviors.

Ethanol affects the function of a neurotransmitter receptor protein without altering the membrane lipid phase.

Using patch-clamp and fluorescence techniques we found that ethanol (10-200 mM) potentiated strychnine-sensitive glycine receptors without having detectable effects on lipid order parameters in mouse spinal cord neurons. Hepthanol (0.01-1 mM), in contrast, did not affect the glycine current, but it altered the core and surface of spinal neuron membranes as detected by changes in 1,6-diphenyl-1,3,5-hexatriene (DPH) and Laurdan fluorescence parameters. These findings support the idea that ethanol affects these membrane proteins without changing lipid fluidity.

[Neurosteroids. Neuromodulators of cerebral excitability]. / Neuroesteroides. Neuromoduladores de la excitabilidad cerebral.

Steroids which are produced by the brain are called neurosteroids, and they are able to modulate neurotransmissions: GABAergic; glutamatergic; glycinergic, and cholinergic (nicotine receptor). These effects are of short latency and duration, and do not implicate the cellular genome. The interaction of these neurosteroids with membrane receptors contribute to the regulation of neuronal excitability, and their study has allowed a better understanding of cognitive, hormonal, and epileptic phenomena as well as the development of new drugs with anxiolytic, antidepressive, anesthetic and anti-epileptic effects.

Changes in the properties of developing glycine receptors in cultured mouse spinal neurons.

We studied several neurophysiological properties of in vitro maturing glycine receptors in mouse spinal cord neurons cultured for various times: 3-7 days (early), 10-12 days (intermediate), and 17-24 days (mature), using whole-cell and gramicidin-perforated techniques. The glycine-activated Cl- conductance increased about 6-fold during in vitro development, and the current density increased from 177+/-42 pA/pF in early to 504+/-74 pA/pF in mature neurons. The sensitivity to glycine increased transiently from 39+/-2.8 microM in early neurons to 29+/-1 microM in intermediate neurons. Using whole-cell recordings, we found that ECl did not change during development. With the gramicidin-perforated technique, on the other hand, ECl shifted from -27 to -52 mV with development. Thus, immature neurons were depolarized by the activation of glycine receptors, whereas mature neurons were hyperpolarized. The current decayed (desensitized) during the application of 500 microM glycine. The decay was single exponential and the time constant increased from 2,212+/-139 msec in early neurons to 4,580+/-1,071 msec in mature neurons. Picrotoxin (10 microM) inhibited the current to a larger extent in early neurons (46+/-6% of control), and the sensitivity of these receptors to strychnine (IC50) increased from 23+/-3 nM to 9+/-1 nM in mature neurons. In conclusion, several properties of spinal glycine receptors changed during in vitro neuronal maturation. This indicates that, similar to GABA(A) receptors, the functions of these receptors are developmentally regulated. These changes should affect the excitability of spinal neurons as well as other maturation processes.

Differential intracellular regulation of cortical GABA(A) and spinal glycine receptors in cultured neurons.

Using patch-clamp techniques we studied several aspects of intracellular GABA(A) and glycine Cl- current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl- current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 microM PMA reduced the amplitude of the GABA(A) and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 microM okadaic acid, a phosphatase inhibitor, induced potentiation in both currents. The amplitude of GABA(A) and glycine currents recorded with 1 mM internal CaCl2 and 10 mM EGTA (10 nM free Ca2+) decayed by less than 30% of control. Increasing the CaCl2 concentration to 10 mM (34 microM free Ca2+) induced a transient potentiation of the GABA(A) current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca2+ concentration. Activation of G proteins with internal FAl4- induced an inhibition of the GABA(A) current, but potentiated the amplitude of the strychnine-sensitive Cl- current. These results indicate that GABA(A) and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca2+. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.

Canales para iones en las membranas celulares: algunos aspectos de interés clínico / Ionic channels in cellular membranes: somo aspects of clinical relevance

En el presente artículo se revisan brevemente las principales características de los canales para iones en las membranas celulares, su participación en diversos eventos fisiológicos y fisiopatológicos y se muestran ciertos aspectos de importancia clínica derivados de su estudio

Ethanol modulation of the gamma-aminobutyric acidA- and glycine-activated Cl- current in cultured mouse neurons.

The effects of ethanol on the GABA (gamma-aminobutyric acid)A-activated Cl- current were studied in cultured mouse hippocampal and cortical neurons using whole-cell techniques. Ethanol (0.25-200 mM) reversibly potentiated the current in 68 of the 131 hippocampal neurons examined. Ethanol also potentiated a strychnine-sensitive glycine-activated Cl- current in hippocampal and spinal neurons. Ethanol (40 mM) enhanced the maximal response to GABA without changing the Hill coefficient (1.2) or the affinity of the receptor for GABA (EC50 = 15 vs. 14 microM). We found neurons with distinct sensitivities to ethanol, and even concentrations of 425 and 850 mM further potentiated the response induced by GABA and glycine. Ethanol was able to potentiate the GABAA current even after removing Ca++ from the external solution. The protein kinase C activator phorbol, 12 myristate, 13 acetate inhibited the amplitude of the GABA current by 73 +/- 7% of control; however, 4-alpha-phorbol, 12 myristate, 13 acetate, its inactive analog, had no effects. In addition, 2 min of preapplication of 1 microM phorbol, 12 myristate, 13 acetate reduced the ethanol-potentiation from 140 +/- 8 to 122 +/- 6%. Recordings of GABA- and glycine-activated Cl- currents showed that low concentrations of ethanol can differentially affect these receptors in a single neuron. This suggests that the GABAergic effect of ethanol is not mediated by a nonspecific change and that different mechanisms might account for the potentiation of these two ligand-activated Cl- channels by ethanol. In addition, the absence of saturation with high concentrations suggests that ethanol modulates these receptor-ion channel complexes by acting in several sites, one of which might control the state of receptor phosphorylation.