Cultured Rat Spinal Cord Neurons Research Articles

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses.

The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells.

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.

Protective effect of molecular hydrogen against H 2 O 2 -induced oxidative injury in cultured rat spinal cord neurons

Objective To investigate the protective effect of molecular hydrogen against hydrogen peroxide(H2O2)-induced oxidative injury in primary cultures of rat spinal cord neurons and the related mechanism.Methods Rat spinal cord neurons were cultured for 7days and were randomly assigned to four groups with different treatments-(1)Normal medium(NM)control:treated with NM;(2)Hydrogen-rich medium(HM):treated with HM;(3)NM+H2O2:treated with 100 μmol/L H2O2and 15μmol/L ferrous chloride(FeCl2)after pretreatment with NM for 2h;and(4)HM+H2O2:treated with 100μmol/L H2O2and 15μmol/L FeCl2after pretreatment with HM for 2h.The respective media were changed every 6hin each group,and 12hlater the cells were collected for assays of reactive oxygen species(ROS),hydroxyl radical(HO·), apoptosis,and the expression of glycogen synthase kinase-3β(GSK-3β)and p-GSK-3β.Results Compared with NM,HM significantly reduced the H2O2-induced intracellular production of ROS and HO·in purified neurons(P0.01),significantly decreased the number of apoptotic neurons(P0.01)and expression of caspase-3(P0.01),and significantly promoted phosphorylation of GSK-3β(P0.01).Conclusion Molecular hydrogen has protective effect against H2O2-induced oxidative injury in primary cultured neurons.The mechanisms might be related to reduction of intracellular production of ROS and HO·,inhibition of apoptosis in neurons and promotion of GSK-3βphosphorylation.

Interleukin-6 inhibits voltage-gated sodium channel activity of cultured rat spinal cord neurons.

Interleukin-6 (IL-6) is a pleiotropic proinflammatory cytokine that plays a key role in the injuries and diseases of the central nervous system (CNS). A voltage-gated Na+ channel (VGSC) is essential for the excitability and electrical properties of the neurons. However, there is still limited information on the role of IL-6 in voltage-gated sodium channels. Our study aimed to investigate the effects of IL-6 on Na+ currents in cultured spinal-cord neurons. VGSC currents were activated and recorded using whole-cell patch-clamp technique in the cultured rat spinal cord neurons. The effects of IL-6 concentration and exposure duration were examined. To determine whether any change in the number of channels in the plasma membrane can inhibit IL-6 on VGSC currents, we examined the expression of α1A (SCN1α) subunit mRNA level and protein level in the neurons before and after IL-6 induction using real-time polymerase chain reaction. We verified that IL-6, through a receptor-mediated mechanism, suppressed Na+ currents in a time- and dose-dependent manner, but did not alter the voltage-dependent activation and inactivation. Gp130 was involved in this inhibition. Furthermore, the spike amplitude was also inhibited by IL-6 in the doses that decreased the Na+ currents. VGSC currents are significantly inhibited by IL-6. Our findings reveal that the potential neuroprotection of IL-6 may result from the inhibitory effects on VGSC currents.

Differential Control of Thrombospondin over Synaptic Glycine and AMPA Receptors in Spinal Cord Neurons

Thrombospondin-1 (TSP-1) is a large extracellular matrix protein secreted by astrocytes during development and inflammation. In the developing CNS, TSP-1 is involved in neuronal migration and adhesion, neurite outgrowth, and synaptogenesis. We investigated the effects of TSP-1 on neurons with mature synapses using immunocytochemistry, single-particle tracking, surface biotinylation, and calcium imaging. We show that in cultured rat spinal cord neurons TSP-1 decreased neuronal excitability by reducing the accumulation of excitatory AMPA receptors (AMPARs) and increasing that of inhibitory glycine receptors (GlyRs) in synapses. The effects of TSP-1 on GlyRs were dependent on the activation of excitatory receptors. These changes were abolished by blocking β1-integrins and mimicked by blocking β3-integrins. In the presence of TSP-1, AMPARs were less stabilized at synapses, increasing their lateral diffusion and endocytosis. Interestingly, TSP-1 counteracted the increased neuronal excitability and neuronal death induced by TNFα. These results suggest a role of TSP-1 in controlling the balance between excitation and inhibition which could help the recovery of normal synaptic activity after injury responses.

Inhibition of chloride outward transport by gadolinium in cultured rat spinal cord neurons

Gadolinium is a rare-earth lanthanide metal ion and is used as organic gadolinium complexes in magnetic resonance imaging (MRI). Although gadolinium-based MRI agents are thought to be safe in clinical use, the in vivo release of the toxic free inorganic gadolinium (Gd 3+) has been reported in some patients with kidney disease. In central nervous system neurons, the inhibitory action of GABA is a consequence of relatively hyperpolarized Cl − equilibrium potential ( E Cl), which results from the activity of K +-Cl − co-transporter (KCC). The lanthanide ions are reported to affect GABA A receptors. However, little is known about the effect of Gd 3+ on GABA A receptor function with intact intracellular Cl − concentration. In the present study, we investigated the effect of Gd 3+ on GABA A receptor-mediated currents using gramicidin perforated patch recording method in cultured rat spinal cord neurons. The application of muscimol, a GABA A receptor agonist, caused outward current at a holding potential of −50 mV. Gd 3+ inhibited the muscimol-induced outward current in a concentration-dependent and reversible manner. Gd 3+ inhibited the maximum muscimol response but had no effect on the half-maximum concentration. The Gd 3+ inhibition was accompanied by a depolarizing shift of the reversal potential. The Gd 3+ action was blocked by furosemide, a blocker of both KCC and Na +-K +-Cl − co-transporter (NKCC), but not bumetanide, a specific blocker of NKCC. Gd 3+ failed to inhibit the muscimol-induced outward currents recorded by conventional whole-cell patch-clamp method which cannot retain intact intracellular Cl − concentration. These results suggest that Gd 3+ inhibits a KCC function and gives rise to increase in intracellular Cl − concentration. The reduction of outward chloride transport could be related to the neurotoxic effects of Gd 3+.

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Homeostatic Regulation of Synaptic GlyR Numbers Driven by Lateral Diffusion

In the spinal cord, most inhibitory synapses have a mixed glycine-GABA phenotype. Using a pharmacological approach, we report an NMDAR activity-dependent regulation of the mobility of GlyRs but not GABA(A)Rs at inhibitory synapses in cultured rat spinal cord neurons. The NMDAR-induced decrease in GlyR lateral diffusion was correlated with an increase in receptor cluster number and glycinergic mIPSC amplitude. Changes in GlyR diffusion properties occurred rapidly and before the changes in the number of synaptic receptors. Regulation of synaptic GlyR content occurred without change in the amount of gephyrin. Moreover, NMDAR-dependent regulation of GlyR lateral diffusion required calcium influx and calcium release from stores. Therefore, excitation may increase GlyR levels at synapses by a calcium-mediated increase in postsynaptic GlyR trapping involving regulation of receptor-scaffold interactions. This provides a mechanism for a rapid homeostatic regulation of the inhibitory glycinergic component at mixed glycine-GABA synapses in response to increased NMDA excitatory transmission.

Vasoactive intestinal peptide influences neurite outgrowth in cultured rat spinal cord neurons

Vasoactive intestinal peptide (VIP) is a neuropeptide which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro . For the purpose of clarifying the effect of VIP on spinal cord neurons, we studied the effect of VIP on neurite outgrowth of fetal rat ventral and dorsal portions of spinal cord in cultures. VIP-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had a significant neurite outgrowth at 10-8, 10-6, and 10-4 M. The effect was considered to be concentration dependent. Morphological changes of the dorsal spinal cord cultures (DSCC) remained unchanged by VIP treatment. Because of their close sequence homology with VIP, PHI-27 (peptide, histidylisoleucine amide) and secretin were also examined with the same experimental conditions as was VIP. Both PHI-27 and secretin had neurite promoting effects in VSCC at 10-8 and 10-6 M, respectively. However, there were no neurite promoting effects in DSCC in both of them at any concentrations. VIP had the most potent effect on neurite outgrowth in VSCC, followed by PHI-27, and secretin in their effectiveness concentrations. Our data showing VIP, PHI-27 and secretin have neurotrophic action on VSCC and suggest that a potential therapeutic use of VIP and its related peptides in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis. [Neurol Res 2001; 23: 851-854]

Expressions of nitrotyrosine and TUNEL immunoreactivities in cultured rat spinal cord neurons after exposure to glutamate, nitric oxide, or peroxynitrite.

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanism is not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosine (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number greatly increased at 48 hr in contrast to the vehicle. Pretreatment of cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulation of NT nitration and the apoptotic pathway in cultured rat spinal neurons.

Neomycin blocks substance P-induced calcium entry in cultured rat spinal cord neurons

Substance P (SP) plays an important role in sensitization of spinal cord neurons. In this study, we investigated SP-induced calcium activities in cultured rat spinal cord neurons with confocal laser scanning microscopy. Our results showed that SP increased [Ca2+]i by calcium entry rather than release from intracellular calcium stores. Neomycin (100 μM), an antagonist of phosphatidylinositol 4,5-bisphosphate (PIP2), blocked SP-induced calcium entry. Ca2+-free medium induced capacitative entry, which was significantly potentiated by SP. As activation of SP receptor (NK-1) leads to production of inositol 1,4,5-trisphosphate (IP3) by PIP2 turnover, the results indicates that SP-induced calcium entry and SP-potentiated capacitative calcium entry might be mediated or regulated by IP3/diacylglycerol (DG) pathway.

Hypothalamic Hypocretin (Orexin): Robust Innervation of the Spinal Cord

Hypocretin (orexin) is synthesized by neurons in the lateral hypothalamus and has been reported to increase food intake and regulate the neuroendocrine system. In the present paper, long descending axonal projections that contain hypocretin were found that innervate all levels of the spinal cord from cervical to sacral segments, as studied in mouse, rat, and human spinal cord and not previously described. High densities of axonal innervation are found in regions of the spinal cord related to modulation of sensation and pain, notably in the marginal zone (lamina 1). Innervation of the intermediolateral column and lamina 10 as well as strong innervation of the caudal region of the sacral cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral hypothalamus. Digital-imaging physiological studies with fura-2 detected a rise in intracellular calcium in response to hypocretin in cultured rat spinal cord neurons, indicating that spinal cord neurons express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible roles in feeding and endocrine regulation, the descending hypocretin fiber system may play a role in modulation of sensory input, particularly in regions of the cord related to pain perception and autonomic tone.

The Development of Excitatory Synapses in Cultured Spinal Neurons

Immunohistochemical studies of synapses in the CNS have demonstrated that glutamate receptors (GluRs) are concentrated at postsynaptic sites in vivo and in vitro (Baude et al., 1995). The mechanisms leading to receptor clustering at excitatory synapses are far less understood than those governing acetylcholine receptor accumulation at the neuromuscular junction () or glycine receptor aggregation at central inhibitory synapses (). Using cultured rat spinal cord neurons, we demonstrate that clustering of the AMPA receptor subunit GluR1 is among the earliest events in excitatory synapse formation in vitro, coincident with the onset of miniature EPSCs and in many cases preceding presynaptic vesicle accumulation. Postsynaptic receptor clustering is induced in a highly specific and reiterative pattern, independent of receptor activation, by contact with a subset of axons capable of inducing receptor clusters. The subunit composition of AMPA receptor clusters varied significantly between neurons but was invariant within a given neuron. The presence of either GluR2 or GluR3 was common to all receptor clusters. Neither high-affinity glutamate transporters nor NMDA receptors appeared to be concentrated with AMPA receptor subunits at these excitatory synapses.

Acute or prolonged exposure to 1-aminocyclopropanecarboxylic acid protects spinal neurons against NMDA toxicity

1-Aminocyclopropanecarboxylic acid (ACPC) is a high affinity partial agonist for the glycine binding site within the NMDA receptor complex. Chronic treatment with ACPC in vivo appears to reversibly desensitize the NMDA receptor complex, prompting suggestions that it might provide an effective means of ameliorating degenerative mechanisms mediated through this ligand-gated ion channel. In the present experiments, cultured rat spinal cord neurons were used to further examine the effects of acute and sustained ACPC exposures on N-methyl- d-aspartate (NMDA)-induced neurotoxicity. Cell damage was quantitatively assessed using a tetrazolium salt colorimetric assay. With coincubation, 1 mM ACPC significantly reduced the neuronal cell damage caused by 30 min exposure to 25 or 50 μM concentrations of NMDA, but, in contrast to other competitive and non-competitive NMDA receptor antagonists ( d-(-)-2-amino-5-phosphonovaleric acid (APV), dizocilpine maleate (MK-801) and 7-chlorokynurenic acid (7-CK)), it failed to alter the cell injury induced by 100 μM NMDA. The protective effect of ACPC was competitively abolished by coaddition of glycine, verifying that it was mediated through glycine binding sites. Sustained 20 h exposure to 1 mM ACPC (which was removed 30 min before addition of 25 μM NMDA) also caused cells to be significantly less responsive to the neurotoxic effects of NMDA. Pre-exposure to ACPC for shorter intervals (< 1 h) failed to alter subsequent NMDA toxicity. Acute or sustained exposures to ACPC alone did not affect cell viability. These results support earlier indications that: (1) ACPC provides an effective means of antagonizing excitotoxic phenomena, and (2) sustained exposure to ACPC desensitizes the NMDA receptor complex.

Clustering of Gephyrin at GABAergic but Not Glutamatergic Synapses in Cultured Rat Hippocampal Neurons

The molecular mechanisms underlying the establishment of a postsynaptic receptor mosaic on CNS neurons are poorly understood. One protein thought to be involved is gephyrin, a peripheral membrane protein that binds to the inhibitory glycine receptor and functions in clustering this receptor at synapses in cultured rat spinal cord neurons. We investigated the possible association of gephyrin with synapses in cultured rat hippocampal neurons, where glutamate and GABA but not glycine are the principal transmitters. Gephyrin immunoreactivity was detected in axons as well as dendrites, changing from a predominantly axonal to a more dendritic distribution with time in culture. Gephyrin staining was not distributed uniformly, but always took the form of clusters. Small clusters of gephyrin (0.2 microns 2), present throughout development, were distributed widely and not restricted to synaptic sites. Larger clusters of gephyrin (0.4-10.0 microns 2, sometimes composed of groups of small clusters), which developed in older cells, were localized to a subset of contacts between axons and dendrites. These large clusters were not present at glutamatergic synapses (marked by immunostaining for GluR1), but were closely associated with GABAergic synapses (marked by immunostaining for GABA and glutamic acid decarboxylase). These results, together with previous findings, suggest that gephyrin may function to anchor GABA and glycine receptors, but not glutamate receptors, at postsynaptic sites on central neurons. They also raise the possibility that gephyrin has additional functions, independent of its role at synapses.

A chromaffin cell-derived protein induces the NADPH-diaphorase phenotype in cultured rat spinal cord neurons.

We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk-signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.

The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton

The mechanisms underlying the postsynaptic localization of neurotransmitter receptors are poorly understood. Recently, the peripheral membrane protein gephyrin has been shown to be essential for the formation of inhibitory glycine receptor clusters in cultured rat spinal cord neurons. In vitro gephyrin binds with high affinity to polymerized tubulin. Here, the interaction of gephyrin with different components of the cytoskeleton was investigated in primary cultures of rat spinal neurons. After treatment with alkaloids affecting the cytoskeleton, the morphology of post-synaptic gephyrin clusters was analyzed by confocal immunofluorescence microscopy. Depolymerization of microtubules by demecolcine reduced both the percentage of cells with postsynaptic gephyrin clusters and the number of clusters/cell. The size of the remaining gephyrin clusters was increased whereas their gephyrin density was significantly lower than under control conditions. Depolymerization of microfilaments by cytochalasin D in contrast generated smaller clusters of increased gephyrin density. Demecolcine also dispersed postsynaptic glycine receptor clusters as revealed by immunostaining with a specific monoclonal antibody. These findings support the view that in vivo gephyrin anchors receptor polypeptides to the cytoskeleton by a complex interaction with microtubules and microfilaments.

Acidic and basic fibroblast growth factors enhance neurite outgrowth in cultured rat spinal cord neurons

We have studied neurotrophic effects of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. Cultures treated with aFGF and bFGF significantly enhanced neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. Our data suggest that aFGF and bFGF are potent neurotrophic factors on rat ventral spinal cord neurons in vitro.

Effect of nocodazole and taxol on glycine-evoked currents in cultured rat spinal cord neurons

Effect of nocodazole and taxol on glycine-evoked currents in cultured rat spinal cord neurons

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure

A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding alpha subunit of the inhibitory glycine receptor forms anion-selective channels in phospholipid bilayers. The most frequent events show single-channel conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau o, in the millisecond time range. These properties match those of authentic glycine receptors studied in inside-out patches of cultured rat spinal cord neurons, namely gamma = 27 pS and gamma = 45 pS, and tau o in the millisecond time range. The channel activity of M2GlyR is sequence-specific: 1) a synthetic peptide with the sequence of putative transmembrane segment M1 (M1GlyR), not considered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays distinct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2GlyR) forms cation-selective channels. Further, a four-helix bundle protein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gamma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, but not by an analog of the local anesthetic lidocaine (QX-222), a cation channel blocker. Observed single-channel properties suggest that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M2 could confer anion specificity to the receptor channel.