[Desensitization of glycine mediated currents in frog spinal cord neurons].

Whole-cell patch clamp recordings from isolated frog spinal cord neurons were made to study the desensitization of glycine-mediated currents. The decay phase of current induced by application of glycine (1 mM) could be either monoexponential or biexponential. Monoexponential decays had either tau1 = 1693 +/- 135 ms (n = 8) or tau2 = 364 +/- 42 ms (n = 9) time constants, while biexponential decays had both tau1 and tau2. Currents with fast monoexponential decay (tau2) remained reproducible through the experiment. Currents with slow monoexponential (tau1) and biexponential decay gradually became faster while whole-cell patch clamp recordings were being taken. These results suggest that, at least, two different glycine receptor subtypes are present in frog spinal cord neurons.

Stoichiometry of the human glycine receptor revealed by direct subunit counting.

The subunit stoichiometry of heteromeric glycine-gated channels determines fundamental properties of these key inhibitory neurotransmitter receptors; however, the ratio of α1- to ß-subunits per receptor remains controversial. We used single-molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2ß. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.

Native glycine receptor subtypes and their physiological roles.

The glycine receptor chloride channel (GlyR), a member of the pentameric Cys-loop ion channel receptor family, mediates inhibitory neurotransmission in the spinal cord, brainstem and retina. They are also found presynaptically, where they modulate neurotransmitter release. Functional GlyRs are formed from a total of five subunits (alpha1-alpha4, beta). Although alpha subunits efficiently form homomeric GlyRs in recombinant expression systems, homomeric alpha1, alpha3 and alpha4 GlyRs are weakly expressed in adult neurons. In contrast, alpha2 homomeric GlyRs are abundantly expressed in embryonic neurons, although their numbers decline sharply by adulthood. Numerous lines of biochemical, biophysical, pharmacological and genetic evidence suggest the majority of glycinergic neurotransmission in adults is mediated by heteromeric alpha1beta GlyRs. Immunocytochemical co-localisation experiments suggest the presence of alpha2beta, alpha3beta and alpha4beta GlyRs at synapses in the adult mouse retina. Immunocytochemical and electrophysiological evidence also implicates alpha3beta GlyRs as important mediators of glycinergic inhibitory neurotransmission in nociceptive sensory neuronal circuits in peripheral laminae of the spinal cord dorsal horn. It is yet to be determined why multiple GlyR synaptic subtypes are differentially distributed in these and possibly other locations. The development of pharmacological agents that can discriminate strongly between different beta subunit-containing GlyR isoforms will help to address this issue, and thereby provide important insights into a variety of central nervous system functions including retinal signal processing and spinal pain mechanisms. Finally, agents that selectively potentiate different GlyR isoforms may be useful as therapeutic lead compounds for peripheral inflammatory pain and movement disorders such as spasticity.

Two different modes of action of pentobarbital at glycine receptor channels.

Glycine receptor channels are pentameric ligand-gated ion channels which respond to the binding of inhibitory transmitters by opening of a chloride-selective central pore. Pentobarbital is widely used as an anticonvulsive, hypnotic and anaesthetic drug. In the present study, the interaction between pentobarbital and glycine receptor channels was studied on outside-out patches of human embryonic kidney (HEK) 293 cells expressing alpha(1)beta glycine receptor channels. Currents elicited by 0.03 mM glycine were enhanced by pentobarbital showing potentiation of alpha(1)beta glycine receptor channels. In the presence of 1 mM glycine+pentobarbital (1 and 3 mM), desensitization was faster and the peak current amplitude decreased. After the end of glycine+pentobarbital pulses, off-currents occurred suggestive for a channel block mechanism. Pentobarbital had no agonistic effects at glycine receptor channels.

N-methyl-D-aspartate receptor-coupled glycineB receptors in the pathogenesis and treatment of schizophrenia: a critical review.

Glutamatergic pathways, metabotropic receptors, and ionotropic alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors are all implicated in the etiology and management of schizophrenia. As concerns NMDA receptors, open channel blockers (OCBs) such as phencyclidine (PCP) elicit psychotic symptoms in human subjects. This observation underpins biochemical studies indicating that a deficit in activity at NMDA receptors may be associated with psychotic states. Inasmuch as agonists at the NMDA recognition site are excitotoxic, drugs acting via the co-agonist, glycine(B) (GLY(B)) site are more promising clinical candidates as antipsychotic agents. Glycine (GLY) itself, a further endogenous agonist, D-Serine, and inhibitors of GLY reuptake are active in certain experimental models predictive of antipsychotic properties. Further, in controlled clinical trials, GLY and D-Serine enhance the ability of conventional neuroleptics such as haloperidol to improve cognitive and negative symptoms. Their actions are mimicked by the partial agonist, D-cycloserine (DCS). However, these agents exert little effect alone and may interfere with therapeutic actions of the atypical antipsychotic, clozapine. An important issue in the interpretation of drug actions at GLY(B) sites is their degree of occupation by endogenous GLY and D-Serine - although they are unlikely to be saturated. Further, distinct "subtypes" of GLY(B) site-bearing NMDA receptor may fulfill differential roles in psychotic states Finally, blockade of certain populations of NMDA receptor may be of use in the management of schizophrenia. This article reviews the complex role of GLY(B) sites/NMDA receptors and their endogenous ligands in the pathogenesis and treatment of psychotic states.

Pharmacological evidence for two types of postsynaptic glycinergic receptors on the Mauthner cell of 52-h-old zebrafish larvae.

The presence of homooligomeric and heterooligomeric glycine receptors (GlyRs) on the Mauthner (M) cell in the isolated medulla of 52-h-old zebrafish larvae was investigated by analysis of the effects of picrotoxin on glycine-gated channels and on glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Two functionally different GlyRs have been previously described on the M cell. The effects of picrotoxin on these two GlyRs were first analyzed by measuring the relative change in their total open probability (NP(o)) with picrotoxin concentration. Picrotoxin had no significant effect on the glycine channel with a single conductance level of 40-46 pS. In contrast, picrotoxin application decreased the NP(o) of the GlyR with multiple subconductance levels. On this GlyR, picrotoxin decreased in a concentration-dependent manner the occurrence of the 80- to 86-pS substate (median inhibiting concentration = 0.89 microM) and had no apparent effect on the 40- to 46-pS opening probability. Opening frequency and the mean open times of the 80- to 88-pS main conductance state were reduced in the presence of 10 microM picrotoxin, but their relative weight remained unchanged. These effects of picrotoxin were not voltage dependent. Picrotoxin also modified 40- to 46-pS kinetics. At 100 microM, picrotoxin evoked voltage-independent flickering during channel openings. Short and long mean open times were significantly decreased, whereas the relative proportion of long mean open times was increased. The medium closed time was decreased, whereas medium burst duration was increased. The burst frequency remained unchanged. Spontaneous glycinergic mIPSCs were recorded in the presence of 1 microM tetrodotoxin + 25 microM bicuculline (holding potential = -50 mV). Application of 10 microM picrotoxin did not change the frequency of the synaptic activity, whereas it decreased the amplitude of large mIPSCs. No effect was observed on the time to peak (0.8 ms) or the mean decay time constant (tau(d) = 7.7 ms). Increasing picrotoxin concentration to 100 microM resulted in a decrease of mIPSC frequency (35.6%), amplitude (39.8%), and tau(d) (from 7.7 to 5 ms). These results suggest that these two functionally different GlyRs correspond to alpha1 homooligomeric-like and alpha1/beta-heterooligomeric-like GlyRs, and that both are synaptically activated. Variation in the proportions of GlyR subtypes from one synapse to another could partly account for the broad amplitude distribution of mIPSCs recorded from the zebrafish M cell.

Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells.

In adult spinal neurons inhibitory glycine receptors (GlyR) are localized at postsynaptic membrane specializations underlying glycinergic nerve terminals. The peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic GlyR clusters. Here, we coexpressed GlyR polypeptides and gephyrin in 293 cells and observed rerouting of hetero-oligomeric GlyR and its beta, but not of alpha subunits to intracellular gephyrin aggregates. A GlyR chimeric alpha 1/beta protein was also accumulated at these gephyrin aggregates, indicating that colocalization with gephyrin depends on cytoplasmic domains of the beta subunit. gamma-Aminobutyric acid type-A receptor (GABAAR) subunits were not targeted to intracellular gephyrin aggregates with the exception of the GABAAR beta 3 subunit, which partially colocalized with gephyrin. These data show that gephyrin alters the subcellular localization of the GlyR beta and, to some extent, GABAAR beta 3 subunits. Thus, gephyrin-binding subunits might target hetero-oligomeric ion channels to a gephyrin matrix underlying the differentiating postsynaptic membrane.

Evolutionary history of the ligand-gated ion-channel superfamily of receptors.

The fast-acting ligand-gated ion channels (LGICs) constitute a group that encompasses nicotinic ACh, 5-HT3, GABAA and glycine receptors. Undoubtedly, they all share a common evolutionary ancestor, and the group can therefore be considered to be a gene superfamily. Because the members of the superfamily are all receptors, it is reasonable to suppose that their common ancestor must also have been some type of receptor, and because the receptors are made of similar subunits, the ancestor was probably homo-oligomeric. Although we failed to find a group of proteins that are related evolutionarily to this superfamily, the analysis of the evolutionary relationships within the superfamily is possible and can give rise to information about the evolution of the structure and function of present-day receptors and indeed of the nervous system itself.