Glycine Channels Research Articles

Quercetin Exerts Anti-convulsant Effects in Animal Model of Grand Mal Epilepsy: Modulation of GABA and Glycinergic Pathways

Quercetin is low molecular weight flavonoid having multiple neuropharmacological actions. Recently, its anticonvulsant effect was reported in rats using electrical kindling models. However, earlier cell culture studies have documented antagonistic action of quercetin on GABAA, GABAC and Glycine channels, which is contrary to recent findings. Hence, the present study aimed at characterizing the effects of quercetin administration in various experimental models of epilepsy. Dose-dependent effects of quercetin on maximal-electroshock seizure (MES) were determined. Further, the effective dose was tested in pentylenetetrazol-induced seizures (PTZ), and strychnine-induced seizures to study the involvement of GABA and Glycinergic receptors. The results revealed a potent anticonvulsant effect of quercetin in MES induced seizures at a dose of 5 and 10 mg/kg. This effect was found to be retained in case of PTZ-induced convulsions and strychnine-induced convulsions (quercetin 10 mg/kg). The present finding warrants further substantiation along with their correlation to molecular mechanism of action.

Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study.

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼ 102 (∼ 20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.

Activation Mechanism of Elic by Propylamine

The pentameric ligand-gated channel ELIC has provided us with high resolution structures of its closed state. This prokaryotic channel opens to a high single-channel conductance in response to a variety of amine compounds. Here we report our tests of kinetic models for the activation of recombinant ELIC channels. Outside-out single channel currents elicited by the full agonist propylamine (0.5-50 mM) were analysed by maximum likelihood direct global fitting of kinetic schemes (HJCFIT program; Colquhoun et al J Physiol 547, 699, 2003). The adequacy of a scheme was judged by comparing the predictions of the best fit obtained for each, with the experimental open/shut time distributions and with the time course of macroscopic propylamine-activated currents (fast theta-tube applications, 50-600 ms, 1-50 mM).Other Cys-loop channels, such as glycine receptors, activate via a pre-opening intermediate ('flip' model, Burzomato et al., J Neurosci 24, 10924,2004) and, when fully liganded, open with high efficacy to a single open state. In contrast with that, ELIC open time distributions at saturating propylamine showed more than one component. Thus, more than one open state must be accessible to the fully liganded channel. The ¯primed¯ model (Mukhtasimova et al., Nature 459, 451, 2009) accounts for that by allowing the channel to open from more than one fully liganded intermediate. The best fit of this type of model showed that ELIC maximum open probability (99%) is reached when three molecules of agonist are bound. The overall efficacy of the fully liganded branch was about 130 (c.f. 20 for α1β glycine channels; Burzomato et al., 2004).

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.

The α1K276E Startle Disease Mutation Reveals Multiple Intermediate States in the Gating of Glycine Receptors

Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2-M3 loop of the glycine receptor α1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK293 cells showed the mutation reduced the receptor glycine sensitivity. In single-channel recordings, rat homomeric α1 K276E receptors were barely active, even at 200 mM glycine. Coexpression of the β subunit partially rescued channel function. Heteromeric mutant channels opened in brief bursts at 300 μM glycine (a concentration that is near-maximal for wild type) and reached a maximum one-channel open probability of about 45% at 100 mm glycine (compared to 96% for wild type). Distributions of apparent open times contained more than one component in high glycine and, therefore, could not be described by mechanisms with only one fully liganded open state. Fits to the data were much better with mechanisms in which opening can also occur from more than one fully liganded intermediate (e.g., "primed" models). Brief pulses of glycine (∼3 ms, 30 mM) applied to mutant channels in outside-out patches activated currents with a slower rise time (1.5 ms) than those of wild-type channels (0.2 ms) and a much faster decay. These features were predicted reasonably well by the mechanisms obtained from fitting single-channel data. Our results show that, by slowing and impairing channel gating, the K276E mutation facilitates the detection of closed reaction intermediates in the activation pathway of glycine channels.

ELIC Channels Activate Slowly in Response to Agonist Concentration Jumps

Prokaryotic members of the Cys-loop superfamily of ligand-gated channels, such as GLIC and ELIC, have given us X-ray structures of different functional states of the channel. Zimmermann & Dutzler (PLOS Biology, 9, e1001101, 2011) have recently shown that ELIC is activated by a range of primary amines, including GABA, making this channel an attractive model for investigating agonist mechanisms. As a first characterisation of ELIC channel kinetics, we have used the technique of rapid theta-tube application to elicit macroscopic agonist currents. Outside-out patches from HEK293 cells expressing ELIC were held at −100 mV and agonists were applied by concentration jumps, 400-550 ms in duration, with exchange times faster than 0.4 ms (20-80%, measured with diluted extracellular solution after patch rupture). We tested propylamine (10-50 mM), cysteamine (10-50 mM) and GABA. For propylamine and cysteamine, the speed with which the current on-relaxation developed reached a clear maximum at 50 mM. At this concentration the time constant for the current risetime was 5.4 +/- 0.75 ms (n = 6) and 7.3 +/- 0.82 ms (n = 9) for cysteamine and propylamine, respectively. Thus, the most striking property of agonist ELIC responses was their slow time course, when compared with mammalian Cys-loop channels, such as the ACh muscle nicotinic or the glycine channel. In similar experiments with glycine channels, the response risetime was effectively too fast to be characterised by this technique, as its time constant reached sub-millisecond values at 3 mM glycine.

The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates (“flip”)

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 µM), openings occurred in long (>300-ms) groups with high open probability (Popen; 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5–10 µs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ∼130,000 s−1, much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum Popen is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.

What single‐channel analysis tells us of the activation mechanism of ligand‐gated channels: the case of the glycine receptor

Glycine receptors are, in several ways, the member of the nicotinic superfamily that is best-suited for single-channel recording. That means that they are ideal for testing ideas about how activation proceeds in a ligand-gated ion channel from the binding of the agonist to the opening of the channel. This review describes the quantitative characterization by single-channel analysis of a novel activation mechanism for the glycine receptor. The favourable properties of the glycine receptor allowed the first detection of a conformation change that follows the binding of the agonist but precedes the opening of the channel. We used the term 'flipping' to describe this pre-opening conformational change. The 'flipped' state has a binding affinity higher than the resting state, but lower than the open state. This increased affinity presumably reflects a structural change near the agonist binding site, possibly the 'capping' of the C-loop. The significance of the 'flip' activation mechanism goes beyond understanding the behaviour and the structure-function relation of glycine channels, as this mechanism can be applied also to other members of the superfamily, such as the muscle nicotinic receptor. The 'flip' mechanism has thrown light on the question of why partial agonists are not efficacious at keeping the channel open, a question that is fundamental to rational drug design. In both muscle nicotinic and glycine receptors, partial agonists are as good as full agonists at opening the channel once flipping has occurred, but are not as effective as full agonists in eliciting this early conformational change.

Conformation Changes Before Opening And The Activation Mechanism In Glycine And Nicotinic Receptors

Channels in the nicotinic superfamily are pentameric membrane proteins that respond to the binding of transmitter molecules to their extracellular domain by opening their integral membrane pore. One of the best ways to obtain information on the chain of events that follows transmitter binding is by single channel analysis. Mechanisms of receptor activation can be fitted to sets of experimental recordings for the purpose of validating a particular model and quantifying the rate at which each step occurs. We use HJCfit, a program developed by David Colquhoun (available from http://www.ucl.ac.uk/pharmacology/dc.html) to obtain maximum-likelihood, global mechanism fits with full missed event correction to steady-state recordings obtained at different agonist concentrations and idealised by time-course fitting. By the use of this technique on wild-type glycine receptors, we were able for the first time to detect an intermediate conformational change that follows agonist binding but precedes channel opening. The short-lived, partially-activated intermediate shut state (which we termed “flip”) has a higher affinity for the agonist than the resting state, which suggests that this conformational change involves some degree of domain closure in the extracellular domain. Activation models that include this flipped state can also accurately describe the properties of ACh nicotinic receptors and of startle disease mutants of the glycine channel. Analysis of the activation of nicotinic channels and glycine channels by partial agonists showed that the difference between partial and full agonists resides in the first conformational change (flipping) rather than in the open-shut reaction as has always been supposed previously. Partial agonists are poor at eliciting the change from resting to flipped, but once in the flipped state the opening and shutting of the channel is much the same for all agonists.

N-Methyl-d-aspartate (NMDA) Receptor Subunit NR1 Forms the Substrate for Oligomeric Assembly of the NMDA Receptor

The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.

Single‐channel properties of glycine receptors of juvenile rat spinal motoneurones in vitro

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 α1β 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 α1β receptors.

A Single P-loop Glutamate Point Mutation to either Lysine or Arginine Switches the Cation–Anion Selectivity of the CNGA2 Channel

Cyclic nucleotide-gated (CNG) channels play a critical role in olfactory and visual transduction. Site-directed mutagenesis and inside-out patch-clamp recordings were used to investigate ion permeation and selectivity in two mutant homomeric rat olfactory CNGA2 channels expressed in HEK293 cells. A single point mutation of the negatively charged pore loop (P-loop) glutamate (E342) to either a positively charged lysine or arginine resulted in functional channels, which consistently responded to cGMP, although the currents were generally extremely small. The concentration–response curve of the lysine mutant channel was very similar to that of wild-type (WT) channels, suggesting no major structural alteration to the mutant channels. Reversal potential measurements, during cytoplasmic NaCl dilutions, showed that the lysine and the arginine mutations switched the selectivity of the channel from cations (PCl/PNa = 0.07 [WT]) to anions (PCl/PNa = 14 [Lys] or 10 [Arg]). Relative anion permeability sequences for the two mutant channels, measured with bi-ionic substitutions, were NO3 − > I− > Br− > Cl− > F− > acetate−, the same as those obtained for anion-selective GABA and glycine channels. The mutant channels also seem to have an extremely small single-channel conductance, measured using noise analysis of about 1–2 pS, compared to a WT value of about 29 pS. The results showed that it is predominantly the charge of the E342 residue in the P-loop, rather than the pore helix dipoles, which controls the cation–anion selectivity of this channel. However, the outward rectification displayed by both mutant channels in symmetrical NaCl solutions suggests that the negative ends of the pore helix dipoles may play a role in reducing the outward movement of Cl− ions through these anion-selective channels. These results have potential implications for the determinants of anion–cation selectivity in the large family of P-loop–containing channels.

Binding site stoichiometry and the effects of phosphorylation on human alpha1 homomeric glycine receptors.

The kinetic properties of the human alpha1 homomeric glycine receptor were investigated. Receptors were expressed in HEK 293 cells, and glycine was applied to outside-out membrane patches with sub-millisecond solution exchange. The activation time course of the glycine response was used to investigate receptor stoichiometry. The unbinding of three strychnine molecules and the cooperative binding of two glycine molecules were required to activate the channel. The effects of phosphorylation on glycine receptor kinetics were investigated by pretreating cells with phosphorylators or with phosphatases. Phosphorylation accelerated desensitisation, but slowed deactivation and recovery from desensitisation. A chemical-kinetic model was developed that reproduced the experimental observations. The model suggests that only three binding sites on the glycine channel are functional, while the remaining two binding sites are 'silent', possibly due to strong negative cooperativity.

Modulation of Baseline Potassium Channels in Cerebellar Granule Neurons of Sprague-Dawley Rats by Sevoflurane

Background: Volatile general anesthetics have been widely used to produce reversible unconsciousness and analgesia in clinical practice over the last one hundred years, but the basic mechanism of anesthetic action is not yet completely understood. In addition to the well known mechanism of and glycine channels, accumulating evidence indicates that neuronal baseline channels are also activated by volatile anesthetics. The goal of this study was to test the hypothesis that sevoflurane, one of the newly developed volatile anesthetics, activates baseline potassium channneis in the cerebellar granule neurons of rats. Methods: Whole cell measurement techniques were performed from cultured cerebellar granule neurons of seven day old male Sprague-Dawley rats using patch clamp techniques to see the effects of two MACs of sevoflurane on baseline channels. Holding potentials were set to -20 mV and collect pulses from - 90 to 90 in 10m V increments of 300 ms duration. The electrode filling solution contained (in mM) 150 KOH, 105 aspartic acid, 3 NaCl, 10 HEPES, 86 glucose, 1 EGTA, 5 (pH 7.4) and standard saline were used as bath solution. The bath contained 150 NaCI, 3 KCI, 10 HEPES, 14 glucose, 1 EGTA, 5 (pH 7.4). Results: Analysis of multiple patch clamp experiments showed the presence of outward-rectifying selective ion channels with a conductance of 1.064 0.32 nS (n = 10) at depolarized potentials. Cerebellar granule neurons exhibit rapid rising, noninactivating, outward-rectifying currents. These channels are insensitive to conventional channel blockers. Clinically relevant concentrations of sevoflurane (518,) increased the baseline K channel outward currents from the control value by 225% in a standard saline perfusate (n = 10, P channels. Activation of baseline channels in central neurons by two MACs of sevoflurane causes membrane hyperpolarization and increases neuronal input conductances providing an additional inhibitory mechanism that could contribute to the overall central depressant effects of this compound.

Attenuation of malonate toxicity in primary mesencephalic cultures using the GABA transport blocker, NO-711.

Cultured rat mesencephalic neurons were used to assess the effects of gamma-aminobutyric acid (GABA) transport blockers on toxicity caused by malonate, a reversible, competitive inhibitor of succinate dehydrogenase. Previous studies utilizing an ex vivo chick retinal preparation have shown that GABA release and cell swelling are early consequences of acute energy impairment and that GABA transport blockers attenuate this toxicity. The present results demonstrate that the nonsubstrate GABA transport blocker, NO-711 (1 nM-1 microM), dose-dependently protected cultured mesencephalic dopamine (DA) and GABA neurons from malonate-induced toxicity. Similar protection was demonstrated with nipecotic acid (1 mM) and SKF89976A (100 nM), substrate and nonsubstrate GABA transport blockers, respectively. These compounds by themselves produced no signs of toxicity, although nipecotic acid caused a long-term decrease in GABA uptake not associated with toxicity. Compounds which decrease intracellular reactive oxygen species (ROS) are protective in this model, but NO-711 did not prevent the rise in intracellular ROS induced by malonate, indicating its protective effects were downstream of ROS production. Supplementation of malonate treated cultures with the GABA(A) agonist, muscimol (10 microM), increased the toxicity toward the DA and GABA neuron populations. Antagonists at the GABA(A) and glycine receptors provided partial protection to both the GABA and DA neurons. These findings suggest that the GABA transporter, GABA(A), and/or glycine channels contribute to cell damage associated with energy impairment in this model.

Direct Measurement of Specific Membrane Capacitance in Neurons

The specific membrane capacitance ( C m) of a neuron influences synaptic efficacy and determines the speed with which electrical signals propagate along dendrites and unmyelinated axons. The value of this important parameter remains controversial. In this study, C m was estimated for the somatic membrane of cortical pyramidal neurons, spinal cord neurons, and hippocampal neurons. A nucleated patch was pulled and a voltage-clamp step was applied. The exponential decay of the capacitative charging current was analyzed to give the total membrane capacitance, which was then divided by the observed surface area of the patch. C m was 0.9 μF/cm 2 for each class of neuron. To test the possibility that membrane proteins may alter C m, embryonic kidney cells (HEK-293) were studied before and after transfection with a plasmid coding for glycine receptor/channels. The value of C m was indistinguishable in untransfected cells and in transfected cells expressing a high level of glycine channels, indicating that differences in transmembrane protein content do not significantly affect C m. Thus, to a first approximation, C m may be treated as a “biological constant” across many classes of neuron.

Cocaine decreases the glycine-induced Cl − current of acutely dissociated rat hippocampal neurons

The effects of cocaine on glycine-induced Cl − current ( I GLY) of single neurons, freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Cocaine depressed I GLY in a concentration-dependent manner, with an IC 50 of 0.78 mM. Preincubation with 1 mM cocaine alone had no effect on I GLY, suggesting that resting glycine channels are insensitive to cocaine. The depression of I GLY by cocaine was independent of membrane voltage. Internal cell dialysis with 1 mM cocaine failed to modify I GLY. Because the depression of I GLY was noncompetitive, cocaine may act on the glycine receptor–chloride ionophore complex at a site distinct from that to which glycine binds. The cocaine suppression of I GLY was unaffected by 1 μM tetrodotoxin and 1 μM strychnine. Blockers of protein kinase C (Chelerythrine), kinase A ( N-[2-(( p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl, (H-89)) and Ca-calmodulin-dependent kinase (1-[ N, O-bis(5-isoquinolinesulfonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62)) were also ineffective, which suggests that these phosphorylating mechanisms do not modulate cocaine-induced suppressant action on I GLY. This extracellular, strychnine-independent depression of I GLY may contribute to cocaine-induced seizures.

Multiple picrotoxinin effect on glycine channels in rat hippocampal neurons

The effect of picrotoxinin on glycine-induced chloride currents was studied in dissociated rat hippocampal neuron culture in whole-cell and excised outside-out patches. Picrotoxinin blocked the glycine induced chloride currents. The picrotoxinin effect at 20 μM on glycine dose response relationship suggested a competitive mechanism. However, at 1 mM, the picrotoxinin effect was largely noncompetitive. In excised patches, glycine activated two types of channels distinguished by a difference in conductances. The first group had single channel conductances of around 47 pS and another around 100 pS. Occasionally, both types of channels were found in the same excised patch. Low concentration of picrotoxinin selectively blocked large conductance channels. At higher concentrations of 0.5 to 1 mM, picrotoxinin blocked the small conductance channels by a flickering block. These findings indicate that the whole-cell glycine current in rat hippocampal neurons is mediated by at least two types of channels. The two types of channels have distinct conductance, picrotoxinin sensitivity and different mechanism of picrotoxinin block.

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.

Evolutionary relationship of the ligand-gated ion channels and the avermectin-sensitive, glutamate-gated chloride channels.

Two cDNAs, GluClalpha and GluClbeta, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707-711, 1994). Expression studies in Xenopus oocytes showed that GluClalpha and GluClbeta have pharmacological profiles distinct from the glutamate-gated cation channels as well as the gamma-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClalpha and GluClbeta genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClalpha and GluClbeta form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClalpha and GluClbeta, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClalpha and GluClbeta belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels.