GLRA1 Research Articles

Identification of the microdeletion breakpoint in aGLRA1null allele of Turkish hyperekplexia patients

Hyperekplexia (startle disease) is a hereditary motor disease caused by mutations within the GLRA1 gene (Chr. 5q33.1), which encodes the alpha1 subunit of the inhibitory glycine receptor (GlyR). While most patients are diagnosed with dominant hyperekplexia associated with point mutations within or adjacent to the channel pore, recessive hyperekplexia is less frequent. Here, we report five new pedigrees of recessive hyperekplexia in apparently unrelated families of Kurdish origin associated with a deletion of exons 1-7 of the GLRA1 gene. The deletion was identical in all families, encompassing 329 Kb of genomic sequence. No other known functional genes were involved, indicating that the GLRA1null allele is distinct from the 5q syndrome. Analysis of the DNA sequence flanking the proximal and distal breakpoint revealed no significant homology of sequences immediately adjacent to the breaks. Consensus sites for Toposiomerase II were detected close to the breakpoint compatible with an illegitimate recombination event. No heterozygous carriers of the deletion allele were detected by screening of 500 individuals from the southeastern Mediterranean region belonging to four different ethnic groups. Hence, the identical nature of the breakpoint junction in all patients and carriers suggests a founder mutation in an ethnic population originating from Turkey.

Molybdenum Cofactor Deficiency Presenting as Neonatal Hyperekplexia: A Clinical, Biochemical and Genetic Study

We report a newborn with exaggerated startle reactions and stiffness of neonatal onset, the prototypical signs of hyperekplexia. Startle and flexor spasms, leading to apnoea, did not respond to treatment with clonazepam but did partially to sodium valproate. Molecular analysis of GLRA1 revealed no mutations. The incidental finding of hypouricemia led to a work-up for molybdenum cofactor (MoCo) deficiency; the diagnosis was confirmed by the altered urine chemistries, including elevated urine S-sulphocysteine. Despite persistence of the spasms, clinical or electrographic seizures were never detected before the infant died at age 1 month. In this patient, the concurrence of hyperekplexia and MoCo deficiency was suggestive of impaired gephyrin function. GPH mutational analysis, however, showed no abnormalities. The patient was eventually found to harbour a novel c.1064T > C mutation in exon 8 of the MOCS1 gene. Despite extensive sequence analysis of the gene, the second causative mutation of this recessive trait still awaits identification. MoCo deficiency should be considered in the differential diagnosis of neonatal hyperekplexia, particularly in the instances of refractoriness to clonazepam, an early demise in infancy or the evidence of no mutations in the GLRA1 gene.

Visceral, inflammatory and neuropathic pain in glycine receptor alpha 3-deficient mice

The alpha3-subunit of strychnine-sensitive glycine receptors is an important modulator of the pain-sensitizing effects of spinal prostaglandin prostaglandin E(2). Mice deficient for alpha3-subunit of strychnine-sensitive glycine receptors lack the prostaglandin E(2)-induced inhibition of glycinergic neurotransmission and recover faster from inflammation-induced hyperalgesia. It, however, remains unclear whether alpha3-subunit of strychnine-sensitive glycine receptors plays a role in other pain models involving prostaglandin synthesis, such as chemically induced pain or neuropathic pain. In this paper, we show a reduction of acetic acid-induced writhing responses in the absence of alpha3-subunit of strychnine-sensitive glycine receptors, but no changes in formalin-induced pain. Furthermore, alpha3-subunit of strychnine-sensitive glycine receptors-deficient mice develop normal thermal hyperalgesia and tactile allodynia. Thus, alpha3-subunit of strychnine-sensitive glycine receptors is involved in the modulation of moderate inflammatory acetic acid-induced pain responses, but neither in formalin-induced pain nor in neuropathic pain.

Brain‐type creatine kinase activates neuron‐specific K+‐Cl– co‐transporter KCC2

GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages as a result of the elevated intracellular Cl- concentration ([Cl-]i). This functional switch is primarily attributable to the K+-Cl- co-transporter KCC2, the expression of which is developmentally regulated in neurons. Previously, we reported that KCC2 interacts with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2 and glycine receptor alpha2 subunit, and gramicidin-perforated patch-clamp recordings were performed to measure the glycine reversal potential (Egly), giving an estimate of [Cl-]i. KCC2-expressing cells displayed the expected changes in Egly following alterations in the extracellular K+ concentration ([K+]o) or administration of an inhibitor of KCCs, suggesting that the KCC2 function was being properly assessed. When added into KCC2-expressing cells, dominant-negative CKB induced a depolarizing shift in Egly and reduced the hyperpolarizing shift in Egly seen in response to a lowering of [K+]o compared with wild-type CKB. Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs, shifted Egly in the depolarizing direction. In primary cortical neurons expressing CKB, the GABA reversal potential was also shifted in the depolarizing direction by DNFB. Our findings suggest that, in the cellular micro-environment, CKB activates the KCC2 function.

Functional recovery of glycine receptors in spastic murine model of startle disease

Clinical variability is common in inherited gene defects of the central nervous system in humans and in animal models of human disorders. Here, we used the homozygous spastic (spa) mutant mice, which resemble human hereditary hyperekplexia, to determine the molecular remodeling of the spinal cord through the course of the disease, and develop a model for clinical disparity between littermates. The spa mutation is an insertion of a LINE-1 element in the gene for the beta subunit of the glycine receptor, Glrb. The insertion causes aberrant splicing in the beta subunit of glycine receptor gene with a consequent important reduction of glycine receptors. At young ages, all homozygous spa animals were spastic, showed loss of glycine receptors, increased expression of vesicular glycine/GABA transporter and NMDA receptors, induction of activated caspase3, and preferential loss of glycinergic interneurons consistent with neurotransmitter toxicity model. Those littermates that recovered from symptoms showed strong over-expression of the glycine receptor alpha 1 subunit (Glra1), and increased myelination and synaptic plasticity. Littermates that showed a deteriorating clinical course failed to over-express Glra1, and also showed relative loss of gephyrin (receptor clustering). These molecular changes were associated with a preferential loss of GABAergic interneurons, and extensive motorneuron loss. These data suggest that functional recovery is likely due to expression of homomeric glycine receptors, rescue from excitotoxicity, and subsequent neuronal remodeling. We propose that human patients with hyperekplexia show remodeling similar to that of the recovering spa mice, as human patients also show a lessening of symptoms as a function of age.

Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+‐mediated inhibition

Glycine receptors exhibit a biphasic sensitivity profile in response to Zn2+-mediated modulation, with low Zn2+ concentrations potentiating (< 10 microm), and higher Zn2+ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn2+-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) alpha1 subunit compared to either GlyR alpha2 or alpha3 subtypes. Swapping the divergent histidine (H107) residue in GlyR alpha1, which together with the conserved H109 forms part of an intersubunit Zn2+-binding site, for the equivalent asparagine residue present in GlyR alpha2 and alpha3, reversed this phenotype. Co-expression of heteromeric GlyR alpha1 or alpha2 with the ancillary beta subunit yielded receptors that maintained their distinctive sensitivities to Zn2+ inhibition. However, GlyR alpha2beta heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR alpha2 homomer. Comparative studies to elucidate the specific residue in the beta subunit responsible for this differential sensitivity revealed instead threonine 133 in the alpha1 subunit as a new vital component for Zn2+-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated beta subunit could indeed affect Zn2+-mediated inhibition but only from one side of the intersubunit Zn2+-binding site, equivalent to the GlyR alpha1 H107 face. This strongly suggests that the alpha subunit is responsible for Zn2+-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn2+-binding site where H109 and T133 are located.

Localization of glycine receptor alpha subunits on bipolar and amacrine cells in primate retina

The major inhibitory neurotransmitter glycine is used by about half of the amacrine cells in the retina. Amacrine cells provide synaptic output to bipolar, ganglion, and other amacrine cells. The present study investigated whether different bipolar and amacrine cell types in the primate retina differ with respect to the expression of glycine receptor (GlyR) subtypes. Antibodies specific for the alpha1, alpha2, and alpha3 subunits of the GlyR were combined with immunohistochemical markers for bipolar and amacrine cells and applied to vertical sections of macaque (Macaca fascicularis) and marmoset (Callithrix jacchus) retinae. For all subunits, punctate immunoreactivity was expressed in the inner plexiform layer. The GlyRalpha2 immunoreactive (IR) puncta occur at the highest density, followed by GlyR(alpha)3 and GlyR(alpha)1 IR puncta. Postembedding electron microscopy showed the postsynaptic location of all subunits. Double immunofluorescence demonstrated that the three alpha subunits are clustered at different postsynaptic sites. Two OFF cone bipolar cell types (flat midget and diffuse bipolar DB3), are predominantly associated with the alpha1 subunit. Two ON bipolar cell types, the DB6 and the rod bipolar cell, are predominantly associated with the alpha2 subunit. The glycinergic AII amacrine cell is presynaptic to the alpha1 subunit in the OFF-sublamina, and postsynaptic to the alpha2 subunit in the ON-sublamina. Another putative glycinergic cell, the vesicular glutamate transporter 3 cell, is predominantly presynaptic to the alpha2 subunit. The dopaminergic amacrine cell expresses the alpha3 subunit at a low density.

Deafness‐related plasticity in the inferior colliculus: gene expression profiling following removal of peripheral activity

The inferior colliculus (IC) is a major center of integration in the ascending as well as descending auditory pathways, where both excitatory and inhibitory amino acid neurotransmitters play a key role. When normal input to the auditory system is decreased, the balance between excitation and inhibition in the IC is disturbed. We examined global changes in gene expression in the rat IC 3 and 21 days following bilateral deafening, using Affymetrix GeneChip arrays and focused our analysis on changes in expression of neurotransmission-related genes. Over 1400 probe sets in the Affymetrix Rat Genome U34A Array were identified as genes that were differentially expressed. These genes encoded proteins previously reported to change as a consequence of deafness, such as calbindin, as well as proteins not previously reported to be modulated by deafness, such as clathrin. A subset of 19 differentially expressed genes was further examined using quantitative RT-PCR at 3, 21 and 90 days following deafness. These included several GABA, glycine, glutamate receptor and neuropeptide-related genes. Expression of genes for GABA-A receptor subunits beta2, beta3, and gamma2, plus ionotropic glutamate receptor subunits AMPA 2, AMPA 3, and kainate 2, increased at all three times. Expression of glycine receptor alpha1 initially declined and then later increased, while alpha2 increased sharply at 21 days. Glycine receptor alpha3 increased between 3 and 21 days, but decreased at 90 days. Of the neuropeptide-related genes tested with qRT-PCR, tyrosine hydroxylase decreased approximately 50% at all times tested. Serotonin receptor 2C increased at 3, 21, and 90 days. The 5B serotonin receptor decreased at 3 and 21 days and returned to normal by 90 days. Of the genes tested with qRT-PCR, only glycine receptor alpha2 and serotonin receptor 5B returned to normal levels of expression at 90 days. Changes in GABA receptor beta3, GABA receptor gamma2, glutamate receptor 2/3, enkephalin, and tyrosine hydroxylase were further confirmed using immunocytochemistry.

Effects of GABA Receptor Antagonists on Retinal Glycine Receptors and on Homomeric Glycine Receptor Alpha Subunits

Glycinergic and GABAergic inhibition are juxtaposed at one retinal synaptic layer yet likely perform different functions. These functions have usually been evaluated using receptor antagonists. In examining retinal glycine receptors, we were surprised to find that commonly used concentrations of GABA antagonists blocked significant fractions of the glycine current. In retinal amacrine and ganglion cells, the competitive GABAA receptor antagonists (bicuculline and SR95531) were also competitive GlyR antagonists. Picrotoxinin produced a noncompetitive inhibition of retinal GlyRs. [1,2,5,6-tetrahydropyridine-4-yl] methylphosphinic acid, the GABACR antagonist, did not inhibit glycine receptors. All three GABAA receptor antagonists were competitive inhibitors of homomeric alpha1 or alpha2 GlyRs expressed in human embryonic kidney cells (HEK293) cells. Interestingly, bicuculline was much more effective at alpha2 GlyRs and might be used to separate glycine receptor subtypes. Thus commonly used concentrations of GABA antagonists do not unambiguously differentiate GABA and glycine pathways. Picrotoxinin inhibition of GABAC receptors requires two amino acids in the second transmembrane region (TM2): 2' serine and 6' threonine. Although TM2 regions in GABA and glycine receptors are highly homologous, neither 2' serine nor 6' threonine is essential for picrotoxinin sensitivity in glycine receptors.

Charge selectivity of the Cys‐loop family of ligand‐gated ion channels

The determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels have been studied for more than a decade. The investigations have mainly covered homomeric receptors e.g. the nicotinic acetylcholine receptor alpha7, the glycine receptor alpha1 and the serotonin receptor 5-HT(3A). Only recently, the determinants of charge selectivity of heteromeric receptors have been addressed for the GABA(A) receptor alpha2beta3gamma2. For all receptor subtypes, the selectivity determinants have been located to an intracellular linker between transmembrane domains M1 and M2. Two features of the M1-M2 linker appear to control ion selectivity. A central role for charged amino acid residues in selectivity has been almost universally observed. Furthermore, recent studies point to an important role of the size of the narrowest constriction in the pore. In the present review, these determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels will be discussed in detail.

Serial Analysis of Gene Expression in Methamphetamine‐ and Phencyclidine‐Treated Rodent Cerebral Cortices: Are There Common Mechanisms?

Pharmacological actions of methamphetamine (METH) and phencyclidine (PCP) are different, but both of them can induce similar psychiatric disorders including abuse, intoxication, withdrawal, and psychotic symptoms like those of schizophrenia. These mental disorders are caused not only by their direct pharmacological effects, but also by secondary brain damage containing gene expression changes. In order to broadly grasp these alterations, we used serial analysis of gene expression (SAGE), a transcriptome analysis. We analyzed three cDNA libraries from cerebral cortices of saline (1 mL/kg)-, METH (4 mg/kg)-, or PCP (10 mg/kg)-treated Wistar rats (one hour after i.p. administration). The numbers of total tags were about 50,000 in each library, and approximately 18,000 kinds of tags were identified respectively. From the comparisons of three groups, we found both METH- and PCP-reactive genes. Upregulated genes contained calmodulin 2, stromal cell-derived factor receptor 1, brain-specific angiogenesis inhibitor 1-associated protein 2, ras homologue enriched in brain, basigin and thyrotropin-releasing hormone receptor. Downregulated genes contained lipocalin 2, aldolase A, importin 13, fatty acid binding protein 3, and glycine receptor alpha2 subunit. These data suggest important clues of common molecular basis in METH- and PCP-related psychiatric disorders.

Two novel mutations of the glycine receptor gene in a Taiwanese hyperekplexia family

The authors report a Taiwanese family with autosomal recessive hyperekplexia. Two novel mutations, W96C (from the paternal allele) and R344X (from the maternal allele), which are located in exon 4 and exon 7 of the GLRA1 gene, were identified in this family. A series of electrophysiologic investigations were conducted in one of the probands, and the results suggest that the "startle center" is located subcortically.

Hereditary hyperekplexia caused by novel mutations of GLRA1 in Turkish families

Hyperekplexia, also known as startle disease or stiff-person syndrome, is a neurological condition characterized by neonatal hypertonia and a highly exaggerated startle reflex. Genetic studies have linked mutations in the gene encoding glycine receptor alpha1 (GLRA1) with hereditary hyperekplexia. We analyzed four Turkish families with a history of hyperekplexia. Genomic DNA was obtained from members of these families, and the entire coding sequence of GLRA1 was amplified by PCR followed by the sequencing of PCR products. DNA sequences were analyzed by direct observation using an electropherogram and compared with a published reference sequence. We identified three novel mutations in GLRA1. These included a large deletion removing the first 7 of 9 exons, a single-base deletion in exon 8 that results in protein truncation immediately after the deletion, and a missense mutation in exon 7 causing a tryptophan-to-cysteine change in the first transmembrane domain (M1). These mutant alleles have some distinct features as compared to previously identified GLRA1 mutations. Our data provides further evidence for mutational heterogeneity in GLRA1. The new mutant alleles reported here should advance our understanding of the etiology of hyperekplexia.

Differential agonist sensitivity of glycine receptor alpha2 subunit splice variants.

1. The glycine receptor (GlyR) alpha2A and alpha2B splice variants differ by a dual, adjacent amino acid substitution from alpha2A(V58,T59) to alpha2B(I58,A59) in the N-terminal extracellular domain. 2. Comparing the effects of the GlyR agonists, glycine, beta-alanine and taurine, on the GlyR alpha2 isoforms, revealed a significant increase in potency for all three agonists at the alpha2B variant. 3. The sensitivities of the splice variants to the competitive antagonist, strychnine, and to the biphasic modulator Zn(2+), were comparable. In contrast, the allosteric inhibitor picrotoxin was more potent on GlyR alpha2A compared to GlyR alpha2B receptors. 4. Coexpression of alpha2A or alpha2B subunits with the GlyR beta subunit revealed that the higher agonist potencies observed with the alpha2B homomer were retained for the alpha2Bbeta heteromer. 5. The identical sensitivity to strychnine combined with a reduction in the maximum current induced by the partial agonist taurine at the GlyR alpha2A homomer, suggested that the changed sensitivity to agonists is in accordance with a modulation of agonist efficacy rather than agonist affinity. 6. An effect on agonist efficacy was also supported by using a structural model of the GlyR, localising the region of splice variation to the proposed docking region between GlyR loop 2 and the TM2-3 loop, an area associated with channel activation. 7. The existence of a spasmodic mouse phenotype linked to a GlyR alpha1(A52S) mutation, the equivalent position to the source of the alpha2 splice variation, raises the possibility that the GlyR alpha2 splice variants may be responsible for distinct roles in neuronal function.

Alanine-scanning mutagenesis in the signature disulfide loop of the glycine receptor alpha 1 subunit: critical residues for activation and modulation.

The glycine receptor enables the generation of inhibitory postsynaptic currents at synapses via neurotransmitter-dependent activation. These receptors belong to the ligand-gated ion channel gene superfamily, in which all members are comprised of five subunits, each of which possesses a signature 13-residue disulfide loop (Cys loop) in the extracellular domain. In this study, we used alanine-scanning mutagenesis of the residues between C138 and C152 of the Cys loop of the glycine receptor alpha1 subunit to identify residues critical for receptor activation and allosteric modulation. Mutation of L142, F145, or P146 to alanine produced decreases in the potency, maximal amplitude, and Hill coefficient for currents elicited by glycine and impaired receptor activation by the agonist taurine. These residues, along with D148, are positionally conserved in the family of LGIC subunits. Mutation at several other positions had little or no effect. The inhaled anesthetics halothane and isoflurane potentiate submaximal agonist responses at wild-type receptors, via an allosteric site. The mutations L142A, F145A, P146A, and D148A abolished positive modulation by these anesthetics, in some cases revealing a small inhibitory effect. A molecular model of the glycine receptor alpha1 subunit suggests that the Cys loop is positioned in a region of the receptor at the interface between the extracellular and transmembrane domains and that the critical functional residues identified here lie along the face of a predominantly hydrophobic surface. The present data implicate the Cys loop as an important functional moiety in the process of glycine receptor activation and allosteric regulation by anesthetics.

The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors

Anaesthetic steroids are established positive allosteric modulators of GABAA receptors, but little is known concerning steroid modulation of strychnine-sensitive glycine receptors, the principal mediators of fast, inhibitory neurotransmission in the brain stem and spinal cord. This study compared the modulatory actions of five anaesthetic pregnane steroids and two non-anaesthetic isomers at human recombinant alpha1 glycine and alpha1beta2gamma2L GABAA receptors. Recombinant alpha1 glycine or alpha1beta2gamma2L GABAA receptors were expressed in Xenopus laevis oocytes and agonist-evoked currents recorded under voltage-clamp. Steroid modulation of currents evoked by GABA, or glycine, was quantified by determining the potency (EC50) and maximal effect of the compounds. The anaesthetics minaxolone (EC50=1.3 microM), Org20599 (EC50=1.1 microM) and alphaxalone (EC50=2.2 microM) enhanced currents mediated by GABAA receptors. The anaesthetics also enhanced currents mediated by glycine receptors, although with higher EC50 values (minaxolone 13.1 microM; Org20599=22.9 microM and alphaxalone=27.8 microM). The maximal enhancement (to 780-950% of control) produced by the three steroids acting at the GABAA receptor was similar, but currents evoked by glycine were potentiated with increasing effectiveness by alphaxalone (199%) <Org20599 (525%) <minaxolone (1197%). The anaesthetic isomers, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one (eltanolone) enhanced GABAA receptor-mediated currents with similar potency and efficacy, but only the former enhanced glycine, the latter causing inhibition. The non-anaesthetic steroids 5alpha-pregnan-3beta-ol-20-one and 5beta-pregnan-3beta-ol-20-one modulated neither GABAA, nor glycine, receptors. The data demonstrate that structure-activity relationships for steroid modulation at glycine and GABAA receptors differ. Comparing the EC50 values reported here with free plasma concentrations during steroid-induced anaesthesia indicates that a selective modulation of GABAA receptor activity is likely to occur in vivo.

Magnetic resonance spectroscopy of cerebral cortex is normal in hereditary hyperekplexia due to mutations in the GLRA1 gene.

Excessive startling and stiffness in hereditary hyperekplexia has been attributed to lack of inhibition at either the cortical or brainstem level. Six patients with hereditary hyperekplexia (HH) and a confirmed mutation in the gene encoding the alpha(1) subunit of the glycine receptor (GLRA1) underwent single voxel (1)H magnetic resonance spectroscopy (MRS) of the brainstem and an area of frontal cortex and white matter using a method that allows absolute quantification of metabolites. The results of MRS were within normal limits, although there was a tendency for the neuronal marker N-acetyl aspartate to be reduced in the brainstem of patients compared with that in controls. Thus, we found no evidence to support a deficit in the cerebral cortex in patients with hereditary hyperekplexia due to mutations in the GLRA1 gene.

A Highly Conserved Aspartic Acid Residue in the Signature Disulfide Loop of the α1 Subunit Is a Determinant of Gating in the Glycine Receptor

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission. This gene superfamily includes the nicotinic acetylcholine, GABAA/C, 5-hydroxytryptamine type 3, and glycine receptors. A signature disulfide loop (Cys loop) in the extracellular domain is a structural motif common to all LGIC member subunits. Here we report that a highly conserved aspartic acid residue within the Cys loop at position 148 (Asp-148) of the glycine receptor alpha1 subunit is critical in the process of receptor activation. Mutation of this acidic residue to the basic amino acid lysine produces a large decrease in the potency of glycine, produces a decrease in the Hill slope, and converts taurine from a full agonist to a partial agonist; these data are consistent with a molecular defect in the receptor gating mechanism. Additional mutation of Asp-148 shows that alterations in the EC50 for agonists are dependent upon the charge of the side chain at this position and not molecular volume, polarity, or hydropathy. This study implicates negative charge at position Asp-148 as a critical component of the process in which agonist binding is coupled to channel gating. This finding adds to an emerging body of evidence supporting the involvement of the Cys loop in the gating mechanism of the LGICs.

Conservation within the RIC-3 Gene Family: EFFECTORS OF MAMMALIAN NICOTINIC ACETYLCHOLINE RECEPTOR EXPRESSION

In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates and invertebrates. All members of this family have two transmembrane domains followed by a coiled-coil domain. Expression of the human ric-3 homolog, hric3, like the C. elegans ric-3, enhances C. elegans DEG-3/DES-2, rat alpha 7, and human alpha 7 nAChR-dependent whole-cell current amplitudes in Xenopus leavis oocytes, thus demonstrating functional conservation. However, hric3 also reduces human alpha 4 beta 2 and alpha 3 beta 4 nAChR-dependent whole-cell current amplitudes. Thus, hric3 shows differential effects on human nAChRs unlike the observed uniform effect of ric-3 on C. elegans nAChRs. Moreover, hric3 totally abolished currents evoked by 5-HT3 serotonin receptors, whereas it barely modified alpha1 glycine receptor currents. With this caveat, RIC-3 belongs to a conserved family of genes likely to regulate nAChR-mediated transmission throughout evolution. Analysis of transcripts encoded by the hric3 locus shows that it encodes for multiple transcripts, likely to produce multiple hric3 isoforms, and that hric3 is expressed in neurons and muscles, thus enabling its interactions with nAChRs in vivo.

Reduced expression of the neuron restrictive silencer factor permits transcription of glycine receptor alpha1 subunit in small-cell lung cancer cells.

Small-cell lung cancer (SCLC) cells express various markers of neuronal differentiation associated with deficient activity of the neuron-restricted silencer factor (NRSF). Here, we characterize mechanisms by which NRSF target genes are upregulated in SCLC and their functional consequences for cell survival. Since the glycine receptor (GlyR) alpha1 subunit gene, GLRA1, contains a sequence motif for NRSF binding (NRSE) within its 5' UTR, it served as a cellular surrogate marker for NRSF activity. Expression of GLRA1 in nontransformed cells is largely restricted to cells in the spinal cord, retina and brain stem. In experiments described here, we detected GLRA1 transcripts in three out of four SCLC-derived cell lines and in three of five biopsy samples obtained from SCLCs. In contrast, no GLRA1 transcripts were found in 10 nonmalignant nor 15 non-small-cell lung cancer biopsies. Consistent with this observation, NRSF-mediated suppression of an expression construct harbouring the NRSE of the GLRA1 (GLRA1 NRSE) gene was impaired in three of four 'classic' SCLC cell lines, whereas exogenous overexpression of NRSF in NRSF-deficient SCLC cell lines reconstituted silencing of the reporter plasmid. The level of NRSF transcripts as well as the level of specifically bound NRSF to the NRSE correlated with the level of GLRA1 transcripts in SCLC cell lines. Splice variants encoding truncated NRSF proteins and expressed in some SCLC did not antagonize the repression of NRSE-containing genes. Most interestingly, reconstitution of NRSF expression induced apoptosis in SCLC cells, suggesting that inhibition of NRSF activity is a crucial step in the carcinogenesis of a subgroup of SCLC.