We found that caffeine is a structural analogue of strychnine and a competitive antagonist at ionotropic glycine receptors (GlyRs). Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes have differing caffeine sensitivity. Tested against the EC(50) of each GlyR subtype, the order of caffeine potency (IC(50)) is: alpha2beta (248 +/- 32 microm) alpha3beta (255 +/- 16 microm) > alpha4beta (517 +/- 50 microm) > alpha1beta(837 +/- 132 microm). However, because the alpha3beta GlyR is more than 3-fold less sensitive to glycine than any of the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine indicate that amphibian retinal ganglion cells do not express a plethora of GlyR subtypes and are dominated by the alpha1beta GlyR. Comparing the effects of caffeine on glycinergic spontaneous and evoked IPSCs indicates that evoked release elevates the glycine concentration at some synapses whereas summation elicits evoked IPSCs at other synapses. Caffeine serves to identify the pharmacophore of strychnine and produces near-complete inhibition of glycine receptors at concentrations commonly employed to stimulate ryanodine receptors.

Glycine receptors (GlyRs) are found in most areas of the brain, and their dysfunction can cause severe neurological disorders. While traditionally thought of as inhibitory receptors, presynaptic-acting GlyRs (preGlyRs) can also facilitate glutamate release under certain circumstances, although the underlying molecular mechanisms are unknown. In the current study, we sought to better understand the role of GlyRs in the facilitation of excitatory neurotransmitter release in mouse visual cortex. Using whole-cell recordings, we found that preGlyRs facilitate glutamate release in developing, but not adult, visual cortex. The glycinergic enhancement of neurotransmitter release in early development depends on the high intracellular to extracellular Cl(-) gradient maintained by the Na(+)-K(+)-2Cl(-) cotransporter and requires Ca(2+) entry through voltage-gated Ca(2+) channels. The glycine transporter 1, localized to glial cells, regulates extracellular glycine concentration and the activation of these preGlyRs. Our findings demonstrate a developmentally regulated mechanism for controlling excitatory neurotransmitter release in the neocortex.

Many pentameric ligand-gated ion channels are modulated by extracellular pH. Glycine receptors (GlyRs) share this property, but it is not well understood how they are affected by pH changes. Whole cell experiments on HEK293 cells expressing zebrafish homomeric α1 GlyR confirmed previous reports that acidic pH (6.4) reduces GlyR sensitivity to glycine, whereas alkaline pH (8.4) has small or negligible effects. In addition to that, at pH 6.4 we observed a reduction in the maximum responses to the partial agonists β-alanine and taurine relative to the full agonist glycine. In cell-attached single-channel recording, low pH reduced agonist efficacy, as the maximum open probability decreased from 0.97, 0.91 and 0.66 to 0.93, 0.57 and 0.34 for glycine, β-alanine and taurine, respectively, reflecting a threefold decrease in efficacy equilibrium constants for all three agonists. We also tested the effect of pH 6.4 in conditions that replicate those at the native synapse, recording outside-out currents elicited by fast application of millisecond pulses of agonists on α1 and α1β GlyR, at a range of intracellular chloride concentrations. Acidic pH reduced the area under the curve of the currents, by reducing peak amplitude, slowing activation and speeding deactivation. Our results show that acidification of the extracellular pH by one unit, as may occur in pathological conditions such as ischaemia, impairs GlyR gating and is likely to reduce the effectiveness of glycinergic synaptic inhibition. KEY POINTS: Extracellular pH in the central nervous system (CNS) is known to shift towards acidic values during pathophysiological conditions such as ischaemia and seizures. Acidic extracellular pH is known to affect GABAergic inhibitory synapses, but its effect on signals mediated by glycine receptors (GlyR) is not well characterised. Moderate acidic conditions (pH 6.4) reduce the maximum single channel open probability of recombinant homomeric GlyRs produced by the neurotransmitter glycine or other agonists, such as β-alanine and taurine. When glycine was applied with a piezoelectric stepper to outside out patches, to simulate its fast rise and short duration at the synapse, responses became shorter and smaller at pH 6.4. The effect was also observed with physiologically low intracellular chloride and in mammalian heteromeric GlyRs. This suggests that acidic pH is likely to reduce the strength of inhibitory signalling at glycinergic synapses.

While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 μM taurine or 10 μM glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μM 4-aminopyridine in low Mg(2+) solution. This proconvulsive effect was prevented by 3 μM strychnine or after incubation with the loop diuretic bumetanide (10 μM), suggesting that it required glycine receptors and an active NKCC1-dependent Cl(-) accumulation. Application of higher doses of taurine (≥ 1 mM) or glycine (100 μM) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μM) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.

An essential step in understanding fast synaptic transmission is to establish the activation mechanism of synaptic receptors. The purpose of this work was to extend our detailed single-channel kinetic characterization of alpha1beta glycine channels from rat recombinant receptors to native channels from juvenile (postnatal day 12-16) rat spinal cord slices. In cell-attached patches from ventral horn neurones, 1 mM glycine elicited clusters of channel openings to a single conductance level (41 +/- 1 pS, n = 12). This is similar to that of recombinant heteromers. However, fewer than 1 in 100 cell-attached patches from spinal neurones contained glycine channels. Outside-out patches gave a much higher success rate, but glycine channels recorded in this configuration appeared different, in that clusters opened to three conductance levels (28 +/- 2, 38 +/- 1 and 46 +/- 1 pS, n = 7, one level per cluster, all levels being detected in each patch). Furthermore, open period properties were different for the different conductances. As a consequence of this, the only recordings suitable for kinetic analysis were the cell-attached ones. Low channel density precluded recording at glycine concentrations other than 1 mM, but the 1 mm data allowed us to estimate the fully bound gating constants by global model fitting of the 'flip' mechanism of Burzomato and co-workers. Our results suggest that glycine receptors on ventral horn neurones in the juvenile rat are heteromers and have fast gating, similar to that of recombinant alpha1beta receptors.

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic, that has a startle phenotype, has a point mutation (A52S) in the glycine receptor alpha1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell-attached patch-clamp recording of single-channel currents elicited by 30-10000 microM glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wild-type receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the 'flip' mechanism, suggests that preopening isomerization to the flipped conformation that follows binding is less favoured in mutant than in wild-type receptors, and, especially, that the flipped conformation has a 100-fold lower affinity for glycine than in wild-type receptors. In contrast, the efficacy of the gating reaction was similar to that of wild-type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC50 for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice.

1. In acutely isolated rat sacral dorsal commisural nucleus (SDCN) neurones, application of kainate (KA) reversibly potentiated glycine-evoked Cl- currents (IGly) in a concentration-dependent manner. 2. The cellular events underlying the interaction between non-NMDA receptors and glycine receptors were studied by using nystatin-perforated patch and cell-attached single-channel recording modes. 3. The action of KA was not accompanied by a shift in the reversal potential for IGly. In dose-response curves, KA potentiated IGly without significantly changing glycine binding affinity. 4. GYKI 52466 blocked while NS-102 had no effect on the KA-induced potentiation of IGly. 5. The potentiation was reduced when KA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. 6. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the action of KA. Inhibition of calcineurin converted the KA-induced potentiation to a sustained one. 7. Single-channel recordings revealed that KA decreased the mean closing time of glycine-gated single-channel activity, resulting in an increase in the probability of channel opening. 8. It is proposed that Ca2+ entry through AMPA receptors modulates the glycine receptor function via coactivation of CaMKII and calcineurin in SDCN neurones. This interaction may provide a new postsynaptic mechanism for control of inhibitory synaptic signalling and represent one of the important regulatory mechanisms of spinal nociception.

1. Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single-channel properties of receptors that contain a mutation, alpha1(K276E), associated with an atypical form of hyperekplexia. 2. Equilibrium concentration-response curves showed that recombinant human alpha1(K276E)beta receptors had a 29-fold lower glycine sensitivity than wild-type alpha1beta receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3. Both wild-type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4. Channel openings were shorter for the alpha1(K276E)beta mutant than for the wild-type alpha1beta, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5. The main effect of the alpha1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose-response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single-channel bursts, and the lack of an effect of the mutation on the strychnine-binding site.

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.

We investigated the properties of glycine receptors and glycinergic synaptic inputs at the axon terminals of rod bipolar cells (RBCs) in rats by patch-clamp recording. Glycine currents recorded from isolated axon terminals were larger than those from isolated somata/dendrites; this was confirmed by puffing glycine onto these two regions in retinal slices. The current density at terminal endings was more than one order of magnitude higher than the density at somatic/dendritic regions. Glycine currents from isolated terminals and isolated somata/dendrites showed similar sensitivity to picrotoxinin blockade. Single-channel opening recorded from isolated terminals and somata/dendrites displayed a similar main-state conductance of ~46 pS. Application of glycine effectively suppressed depolarization-evoked increases in intracellular Ca2+ at the terminals. In the presence of GABAA and GABAC antagonists, strychnine-sensitive chloride currents were evoked in RBCs in retinal slices by puffing kainate onto the inner plexiform layer. No such currents were observed if the recorded RBCs did not retain axon terminals or if Ca2+ was replaced by Co2+ in the extracellular solution. The currents displayed discrete miniature-like events, which were partially blocked by tetrodotoxin. Consistent with early studies in the rabbit and mouse, this study demonstrates that glycine receptors are highly concentrated at the axon terminals of rat RBCs. The pharmacological and physiological properties of glycine receptors located in the axon terminal and somatic/dendritic regions, however, appear to be the same. This study provides evidence for the existence of functional glycinergic synaptic input at the axon terminals of RBCs, suggesting that glycine receptors may play a role in modulating bipolar cell synaptic transmission.

The physiological and pharmacological properties of taurine-induced responses were investigated in dopaminergic (DA) neurones from the ventral tegmental area (VTA) of young rats aged 1-13 postnatal days, either in acute brain slices or acutely dissociated neurones. When whole-cell responses were recorded from current-clamped neurones using the gramicidin-perforated technique, the application of taurine (0.01-30 mm) accelerated firings and induced membrane depolarization. In voltage-clamped neurones, taurine induced a current which was antagonized by strychnine and by picrotoxin, but not by bicuculline. In addition, taurine-induced current showed complete cross-desensitization with glycine-activated currents but not with gamma-aminobutyric acid (GABA)-activated currents. Thus, taurine is a full agonist of the glycine receptors (GlyRs) in the VTA. Further studies found that taurine acted mainly on non-synaptic GlyRs. The application of 20 microm bicuculline abolished the spontaneous inhibitory post-synaptic currents (IPSCs) in 40/45 neurones, and 93% of the evoked IPSCs. The addition of 1 microm strychnine completely eliminated the remaining IPSCs. These results suggest that GABAergic IPSCs predominate, and that functional glycinergic synapses are present in a subset of the VTA neurones. The application of 1 mum strychnine alone induced an outward current, suggesting that these neurones were exposed to tonically released taurine/glycine. In conclusion, by activating non-synaptic GlyRs, taurine may act as an excitatory extra-synaptic neurotransmitter in the VTA during early development.

AII amacrine cells play a crucial role in retinal signal transmission under scotopic conditions. We have used rat retinal slices to investigate the functional properties of inhibitory glycine receptors on AII cells by recording spontaneous IPSCs (spIPSCs) in whole cells and glycine-evoked responses in outside-out patches. Glycinergic spIPSCs displayed fast kinetics with an average 10-90% rise time of approximately 500 mus, and a decay phase best fitted by a double-exponential function with tau(fast) approximately 4.8 ms (97.5% amplitude contribution) and tau(slow) approximately 33 ms. Decay kinetics were voltage dependent. Ultrafast application of brief ( approximately 2-5 ms) pulses of glycine (3 mm) to patches, evoked responses with fast deactivation kinetics best fitted with a double-exponential function with tau(fast) approximately 4.6 ms (85% amplitude contribution) and tau(slow) approximately 17 ms. Double-pulse experiments indicated recovery from desensitization after a 100-ms pulse of glycine with a double-exponential time course (tau(fast) approximately 71 ms and tau(slow) approximately 1713 ms). Non-stationary noise analysis of spIPSCs and patch responses, and directly observed channel gating yielded similar single-channel conductances ( approximately 41 to approximately 47 pS). In addition, single-channel gating occurred at approximately 83 pS. These results suggest that the fast glycinergic spIPSCs in AII cells are probably mediated by alpha1beta heteromeric receptors with a contribution from alpha1 homomeric receptors. We hypothesize that glycinergic synaptic input may target the arboreal dendrites of AII cells, and could serve to shunt excitatory input from rod bipolar cells and transiently uncouple the transcellular current through electrical synapses between AII cells and between AII cells and ON-cone bipolar cells.

The GABA(A) antagonist SR-95531 (gabazine) is known to block glycine receptors, albeit with low affinity. We have studied the effect of SR-95531 on rat recombinant alpha1beta glycine receptors expressed in human embryonic kidney (HEK293) cells by recording macroscopic currents elicited by rapid glycine application to outside-out patches. SR-95531 has a fast unbinding rate (k(offSR), about 3000 s(-1)) and this means that the time course of its unbinding is comparable to the expected time course of the transmitter in the cleft. We also found that equilibrium applications of SR-95531 reduced the response to brief glycine applications by an amount inversely proportional to the duration of glycine application. The fast unbinding rate of SR-95531 from the glycine receptor will make it useful for establishing the time course of glycine concentration at glycinergic synapses.

The functional properties of glycine receptors were analysed in different types of wide-field amacrine cells, narrowly stratifying cells considered to play a role in larger-scale integration across the retina. The patch-clamp technique was used to record spontaneous IPSCs (spIPSCs) and glycine-evoked patch responses from mature rat retinal slices (4-7 weeks postnatal). Glycinergic spIPSCs were blocked reversibly by strychnine (300 nM). Compared to previously described spIPSCs in AII amacrine cells, the spIPSCs in wide-field amacrine cells displayed a very slow decay time course (tau(fast) approximately 15 ms; tau(slow) approximately 57 ms). The kinetic properties of spIPSCs in whole-cell recordings were paralleled by even slower deactivation kinetics of responses evoked by brief pulses of glycine (3 mm) to outside-out patches from wide-field amacrine cells (tau(fast) approximately 45 ms; tau(slow) approximately 350 ms). Non-stationary noise analysis of patch responses and spIPSCs yielded similar average single-channel conductances (approximately 31 and approximately 34 pS, respectively). Similar, as well as both lower- and higher-conductance levels could be identified from directly observed single-channel gating during the decay phase of spIPSCs and patch responses. These results suggest that the slow glycinergic spIPSCs in wide-field amacrine cells involve alpha2beta heteromeric receptors. Taken together with previous work, the kinetic properties of glycine receptors in different types of amacrine cells display a considerable range that is probably a direct consequence of differential expression of receptor subunits. Unique kinetic properties are likely to differentially shape the glycinergic input to different types of amacrine cells and thereby contribute to distinct integrative properties among these cells.

Spinal parvalbumin-expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn. This study assessed the inhibitory regulation of the parvalbumin-expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability. Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin-expressing interneuron excitability. Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/β subunit-containing receptors underlie both synaptic and tonic glycinergic currents. Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin-expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing.

The amino acids glycine, beta-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, beta-alanine and taurine upon homomeric alpha1 human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10-90 % rise times determined from rapid application of 100 microM of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 mM) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for beta-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for beta-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > beta-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor.

1. Whole-cell glycine-activated currents were recorded from human embryonic kidney (HEK) cells expressing wild-type and mutant recombinant homomeric glycine receptors (GlyRs) to locate the inhibitory binding site for Zn2+ ions on the human alpha1 subunit. 2. Glycine-activated currents were potentiated by low concentrations of Zn2+ (<10 microM) and inhibited by higher concentrations (>100 microM) on wild-type alpha1 subunit GlyRs. 3. Lowering the external pH from 7.4 to 5.4 inhibited the glycine responses in a competitive manner. The inhibition caused by Zn2+ was abolished leaving an overt potentiating effect at 10 microM Zn2+ that was exacerbated at 100 microM Zn2+. 4. The identification of residues involved in the formation of the inhibitory binding site was also assessed using diethylpyrocarbonate (DEPC), which modifies histidines. DEPC (1 mM) abolished Zn2+-induced inhibition and also the potentiation of glycine-activated currents by Zn2+. 5. The reduction in glycine-induced whole-cell currents in the presence of high (100 microM) concentrations of Zn2+ did not increase the rate of glycine receptor desensitisation. 6. Systematic mutation of extracellular histidine residues in the GlyR alpha1 subunit revealed that mutations H107A or H109A completely abolished inhibition of glycine-gated currents by Zn2+. However, mutation of other external histidines, H210, H215 and H419, failed to prevent inhibition by Zn2+ of glycine-gated currents. Thus, H107 and H109 in the extracellular domain of the human GlyR alpha1 subunit are major determinants of the inhibitory Zn2+ binding site. 7. An examination of Zn2+ co-ordination in metalloenzymes revealed that the histidine- hydrophobic residue-histidine motif found to be responsible for binding Zn2+ in the human GlyR alpha1 subunit is also shared by some of these enzymes. Further comparison of the structure and location of this motif with a generic model of the GlyR alpha1 subunit suggests that H107 and H109 participate in the formation of the inhibitory Zn2+ binding site at the apex of a beta sheet in the N-terminal extracellular domain.

Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and β subunits assembled in a 2:3 stoichiometry. The α1/βheteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH.We usedwhole cell recording (at 22-24◦C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg⁻¹, I.P.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic micewere reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P <0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P <0.05; means ± SEM, n =34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 =130 ± 20 μM vs. 64 ± 11 μM, respectively; n =8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDHneurones.Glycine sensitivity, based on the response to 1-3 μM glycine, was reduced in>75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.

Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express alpha2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly alpha1/beta heteromeres). Here, the kinetics properties of GlyRs at the single-channel level have been recorded in real-time by means of the patch-clamp technique in the outside-out configuration coupled with an ultra-fast flow application system (< 100 micros). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the alpha2 GlyR subunit. We show that the onset, the relaxation and the desensitisation of alpha2 homomeric GlyR-mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand-gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mM glycine; 1 ms pulse duration), the opening probability of alpha2 homomeric GlyRs was low (P(o) approximately = 0.1) when compared to mature synaptic GlyRs (Po = 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of alpha2 homomeric GlyRs when compared to the activation kinetics of mature alpha1/beta GlyRs. Such slow kinetics suggest that embryonic alpha2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non-synaptic paracrine-like release of agonist, which is known to occur in the embryo.

Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission. Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem. A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs. These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact. Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease.