Hyperekplexia Research Articles

The glycinergic inhibitory synapse.

Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has yielded new insights on glycine neurotransmission. Glycinergic synapses are now known not to be restricted to the spinal cord and the brainstem. Presynaptic machinery for glycine release and uptake, the structure and function of postsynaptic receptors and the factors (both pre- and postsynaptic) which control the strength of glycinergic inhibition have been extensively studied. It is now established that glycinergic synapses can be excitatory in the immature brain and that some inhibitory synapses can corelease gamma-aminobutyric acid (GABA) and glycine. Moreover, the presence of glycine transporters on glial cells and the capacity of these cells to release glycine suggest that glycine may also act as a neuromodulator. Extensive molecular studies have revealed the presence of distinct subtypes of postsynaptic glycine receptors with different functional properties. Mechanisms of glycine receptors aggregation at postsynaptic sites during development are better understood and functional implications of variation in receptor number between postsynaptic sites are partly elucidated. Mutations of glycine receptor subunits have been shown to underly some human locomotor disorders, including the startle disease. Clearly, recent work on glycine receptor channels and the synapses at which they mediate inhibitory signalling in both young and adult animals necessitates an update of our vision of glycinergic inhibitory transmission.

Genetics, an alternative way to discover, characterize and understand ion channels.

1. The conventional approach to understanding the structure and properties of ion channels has been to use physiological characterization. 2. Purification and molecular cloning of ion channel genes has enabled more detailed structure-function analyses to be undertaken. 3. An alternative approach to the identification of genes of pathophysiological importance has been the use of genetic linkage approaches and positional cloning or positional candidate analysis of ion channel genes. 4. Using genetic approaches, mutations have been described that cause inherited neurological disorders of neurons (e.g. epilepsy, migraine, deafness, ataxia and startle disease), skeletal muscle (myotonia, malignant hyperthermia, periodic paralysis and myasthenia) and cardiac muscle (long QT syndrome and ventricular fibrillation). 5. For each disease, gene structure-function analyses of the mutant alleles have provided further insights into the biology of ion channels. 6. The present brief review examines the methods used in genetic linkage studies and positional cloning of disease genes. Understanding how ion channel gene mutations give rise to dysfunctional channels will be important in defining and treating the episodic and chronic channelopathies.

Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor beta-subunit: an animal model of startle disease.

Startle disease or hereditary hyperekplexia has been shown to result from mutations in the α1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR α or β-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR β transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR β-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR β transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at ∼ 3–4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease.

Structure and functions of inhibitory and excitatory glycine receptors.

The strychnine-sensitive glycine receptor (GlyR) is a pentameric chloride channel protein that exists in several developmentally and regionally regulated isoforms in the CNS. These result from the differential expression of four genes encoding different variants (alpha 1-alpha 4) of the ligand-binding subunit of the GlyR. Their assembly with the structural beta subunit is governed by "assembly cassettes" within the extracellular domains of these proteins and creates chloride channels of distinct conductance properties. GlyR gating is potentiated by Zn2+, a metal ion co-released with different neurotransmitters. Site-directed mutagenesis has unraveled major determinants of agonist binding and Zn2+ potentiation. During development, glycine receptors mediate excitation that results in Ca2+ influx and neurotransmitter release. Ca2+ influx triggered by the activation of embryonic GlyRs is required for the synaptic localization of the GlyR and its anchoring protein gepyhrin. In the adult, mutations in GlyR-subunit genes result in motor disorders. The spastic and spasmodic phenotypes in mouse as well as human hereditary startle disease will be discussed.

The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating.

Hyperekplexia (startle disease) results from mutations in the glycine receptor chloride channel that disrupt inhibitory synaptic transmission. The Q266H missense mutation is the only hyperekplexia mutation located in the transmembrane domains of the receptor. Using recombinant expression and patch-clamping techniques, we have investigated the functional properties of this mutation. The ability of glycine and taurine to open the channel was reduced in the mutated channel, as shown by a 6-fold shift in the concentration-response curve for both agonists. This was not accompanied by similar changes in agonist displacement of strychnine binding, suggesting that the mutation affects functions subsequent to ligand binding. Taurine was also converted to a weak partial agonist and antagonized the actions of glycine, consistent with changes in its channel gating efficacy. Because the Q266H mutation is within the pore-forming second transmembrane domain, we tested for a direct interaction with permeating ions. No change in either the cation/anion selectivity ratio or in single channel conductance levels was observed. No differential effects of Zn++, pH, and diethylpyrocarbonate were observed, implying that the histidine side chain is not exposed to the channel lumen. Single-channel recordings revealed a significant reduction in open times in the mutant receptors, at both high and low agonist concentrations, consistent with the open state of the channel being less stable. This study demonstrates that residues within the second transmembrane domain of ligand-gated ion channel receptors, even those whose side chains do not directly interact with permeating ions, can affect the kinetics of channel gating.

Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes.

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

Late-onset startle syndrome and obsessive compulsive disorder.

A case of late onset sporadic startle syndrome in a patient with a right posterior fossa brain tumour is reported. The exaggerated startle response did not respond to treatment with clonazepam. In addition to anxiety and depression, the patient developed obsessive- compulsive symptoms which responded to behavioural therapy. The possible mechanisms for this unique pattern of symptoms are discussed.

Heritable Mutations in the Glycine, GABAA/ and Nicotinic Acetylcholine Receptors Provide New Insights into The Ligand-Gated Ion Channel Receptor Superfamily

Publisher Summary This chapter highlights new set of information that has become available with the identification of heritable disease-causing mutations in various ligand-gated ion channel (LGIC) receptor subunits. The chapter summarizes the collective state of the structural and functional knowledge of the LGIC receptor superfamily. Comparison of the sites of heritable mutations with existing knowledge of the structure and function of the ligand gated ion channel receptors predicts the likely consequences of some heritable mutations. Whereas others, particularly the mutations that cause hyperekplexia in the glycine receptor, have defined new receptor domains involved in the process of signal transduction. The significance of these findings is that they provide a means of developing a unified model of the mechanism of action of the ligand-gated ion channel receptors. Excitatory signals are mediated by neurotransmitters such as acetylcholine, whereas inhibitory signals are mediated by glycine or γ-aminobutyric acid (GABA). The identification of naturally occurring heritable mutations in the various members of the LGIC receptor superfamily has provided a new set of targets for structure-function analysis. Mutations in subunits of the glycine receptor lead to a variety of startle syndromes in man and mouse. Mutations in subunits of the nicotinic acetylcholine receptor cause several forms of congenital myasthenia gravis and a rare form of epilepsy, whereas GABA A receptor subunit mutations result in insecticide resistance and alcohol sensitivity.

Hyperekplexia-like syndromes without mutations in the GLRA1 gene

Hyperekplexia (MIM: 149400), or startle disease, is an autosomal dominant neurological disorder characterized by an extreme generalized stiffness immediately after birth, normalizing during the first years of life. Other features of this disorder are excessive startle reactions to unexpected, particularly auditory, stimuli together with a short period of generalized stiffness during which voluntary movements are impossible. Linkage analysis mapped a gene for this disorder to chromosome 5833-q35 Subsequently, mutations in the GLRAI gene encoding the α1-subunit of the glycine receptor proved to be causally related to the disease. In the present study, mutation analysis of all exon and flanking intron sequences of this gene was performed in sporadic patients and their parents. Moreover, a branch of the original Dutch hyperekplexia family with a very severely affected individual was screened for an additional mutation in the GLRAI gene. Except for two polymorphisms, of which one results in an amino acid change, no potentially disease causing mutations were found in the α I-subunit of the glycine receptor. Together with haplotype analysis these results exclude a recessive inheritance or new mutation etiology in these hyperekplexia-like syndromes and emphasize that hyperekplexia-like syndromes can be caused by other genetic factors. The involvement of other genes encoding subunits of the functional glycine receptor complex has not been excluded.

The frameshift mutation oscillator ( Glra1 spd-ot) produces a complete loss of glycine receptor α1-polypeptide in mouse central nervous system

Mice homozygous for the recessive mutation oscillator ( Glra1 spd-ot ) suffer from a complex motor disorder leading to death within three weeks after birth. Symptoms of this disorder mimic poisoning by strychnine, the antagonist of the inhibitory glycine receptor. The syndrome has previously been correlated to a 7 base pair microdeletion within the Glra1 gene (chromosome 11) encoding the α1-subunit of the adult glycine receptor isoform. As shown by [ 3H]strychnine binding and western blot analysis employing subunit-specific antibodies, spinal cord of homozygous oscillator mice was totally devoid of α1-polypeptide, characterizing the Glra1 spd-ot gene as a functional null allele of Glra1. Moreover, tissue levels of the postsynaptic anchoring protein gephyrin were drastically reduced in the Glra1 spd-ot/Glra1 spd-ot genotype. In contrast, immunoanalysis revealed a persisting low-level expression of non-α1 glycine receptor polypeptides. Spinal glycine receptor content was also significantly reduced in the +/Glra1 spd-ot genotype. This reduction coincided with increased acoustic startle responses in heterozygous animals consistent with haplotype insufficiency of glycine receptor function. Lethality of the murine null allele Glra1 spd-ot contrasts with the situation in the human, where homozygosity for a GLRA1 null allele produces the phenotype of the non-lethal disorder hyperekplexia (startle disease; stiff baby syndrome). This suggests a disperate regulation of glycine receptor subunit genes and/or diverse compensatory pathways in mice and humans.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel.

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

Startle disease in an Italian family by mutation (K276E): The α‐subunit of the inhibiting glycine receptor

Startle disease in an Italian family by mutation (K276E): The α‐subunit of the inhibiting glycine receptor

Startle disease in an Italian family by mutation (K276E): The alpha-subunit of the inhibiting glycine receptor.

Startle disease in an Italian family by mutation (K276E): The alpha-subunit of the inhibiting glycine receptor.

Increased startle responses in mice carrying mutations of glycine receptor subunit genes.

An exaggerated startle response caused by mutations of the alpha 1 subunit gene of the inhibitory glycine receptor (GLRA1) is the key symptom of human hyperekplexia or startle disease. The recessive mouse mutant spasmodic (spd) carries a missense mutation in the corresponding murine Glra1 gene which reduces the affinity of agonists for the mutant receptor. This mutant has been regarded as an animal model with which to investigate the molecular basis of hyperekplexia and related motor disorders. The recessive mouse mutant spastic (spa) carries an insertional mutation in the glycine receptor beta subunit gene (Glrb) that results in aberrant splicing and, consequently, in a reduced number of functionally intact receptors. The resulting phenotype is similar to that of spasmodic. This study measured the acoustic startle response of spasmodic and spastic mice under different stimulus conditions, in order to test for sensorimotor processing deficits in these animals. Both mutants show increased startle responses to acoustic stimuli of different intensities compared with wild-type animals.

A case of Startle disease with EEG abnormality

A case of Startle disease with EEG abnormality

Mutations Affecting the Glycine Receptor Agonist Transduction Mechanism Convert the Competitive Antagonist, Picrotoxin, into an Allosteric Potentiator

Contrary to its effects on the gamma-aminobutyric acid type A receptor, picrotoxin antagonism of the alpha 1 subunit of the human glycine receptor is shown to be competitive, not use-dependent, and nonselective between the picrotoxin components, picrotin, and picrotoxinin. Competitive antagonism and non-use dependence are consistent with picrotoxin binding to a site in the extracellular domain. The mutations Arg-->Leu or Arg-->Gln at residue 271 of the glycine receptor alpha 1 subunit, which are both associated with human startle disease, have previously been demonstrated to disrupt the transduction process between agonist binding and channel activation. We show here that these mutations also transform picrotoxin from an allosterically acting competitive antagonist to an allosteric potentiator at low (0.01-3 microM) concentrations and to a noncompetitive antagonist at higher (> or = 3 microM) concentrations. This demonstrates that arginine 271 is involved in the transduction process between picrotoxin binding and its mechanism of action. Thus, the allosteric transduction pathways of both agonists and antagonists converge at a common residue prior to the activation gate of the channel, suggesting that this residue may act as an integration point for information from various extracellular ligand binding sites.

Review : Glycine Receptors: Molecular Heterogeneity and Implications for Disease

The inhibitory glycine receptor is a ligand-gated chloride channel that exists in developmentally regulated isoforms. These oligomeric transmembrane proteins are composed of variants of the ligand binding α subunit and structural β polypeptides. The agonist and antagonist sites of the α subunits are formed by discontinuous sequence motifs. In the murine genome, the genes encoding the α1 ( Glra1), α3 ( Glra3), and β ( Glyrb) subunit are autosomally located, whereas the α2 ( Glra2) and α4 ( Glra4) genes reside on the X-chromosome. Mutations of glycine receptor genes have been found to underly hypertonic motor disorders in mice and humans. The mouse mutants spasmodic (spd) and oscillator ( spdot) carry recessive mutations of the Glra 1 gene. In the phenotypically similar mouse mutant spastic ( spa), the intronic insertion of a LINE-1 transposable element into the Gyrb gene results in the aberrant splicing and a consecutive loss of glycine receptors. The human neurological disorder hyperekplexia (startle disease, stiff baby syndrome) is caused by point mutations within the α1 subunit gene ( GLRA1) localized in the human chromosomal region 5q31.3. The Neuroscientist 1:130- 141,1995

Reflex epilepsy in the Papio-papio baboon, particularly photosensitive epilepsy.

Papio-papio baboons may present two types of reflex paroxysmal manifestations: --Myoclonia and generalized seizures are induced by intermittent light stimulation in predisposed animals; this photosensitive epilepsy resembles that observed in some human patients; it involves mainly the cerebral cortex during myoclonia which are accompanied by EEG paroxysmal discharges, and the mesencephalic reticular formation during seizures; --Myoclonia of a different type, never accompanied by EEG paroxysmal discharged and never evolving into seizures, may occur during movement or agitation of predisposed animals; these myoclonia are considered "non-epileptic" since they do not involve the cerebral cortex but probably the lower brain stem; they resemble that observed in startle disease or in some human degenerative disorders. The paper demonstrates that these manifestations constitute two different entities with clinical and electrophysiological characteristics as well as pharmacological reactivities completely different one from the other. Their "epileptic" or "non-epileptic" nature is discussed.

Glycine receptors in the caudal pontine reticular formation: are they important for the inhibition of the acoustic startle response?

The present paper sought to test the hypothesis that inhibitory glycine receptors (GlyRs) on giant neurons of the caudal pontine reticular formation (PnC) are involved in the inhibition of the acoustic startle response (ASR) in rats. First we provided evidence for the presence of the strychnine-sensitive inhibitory GlyR on PnC neurons by immunocytochemical labeling using an antibody against the α 1 subunit of the GlyR. We then measured the ASR as well as two ASR inhibiting phenomena, short-term habituation and prepulse inhibition, after microinjections of the glycine antagonist strychnine (0, 5 or 10 nmol) or the glycine agonist β-alanine (0, 50 or 100 nmol) into the PnC. Neither strychnine nor β-alanine had a measurable influence on any of the parameters of the ASR investigated (amplitude, short-term habituation, prepulse inhibition). In contrast, systemic injection of strychnine (1 mg/kg) markedly increased the ASR amplitude. The systemic administration of strychnine did not impair prepulse inhibition. The human ‘startle disease’ (hyperekplexia), an exaggerated startle response, is caused by a defect of the α 1 subunit of the inhibitory GlyR, but it is unclear at which site in the central nervous system this defect ultimately leads to the symptoms of hyperekplexia. Our data indicate that a blockade of the inhibitory GlyRs in the PnC does not affect the ASR of rats, suggesting that deficient GlyRs in the PnC might not be involved in the etiology of the human ‘startle disease’. We conclude that the inhibitory GlyRs on PnC neurons are not necessary for the inhibition of the ASR and believe that they are involved in another behavioral context.

Molecular mechanisms of inherited startle syndromes

Inherited neurological disorders involving an exaggerated startle response to unexpected sensory stimuli have been identified in mice, cows, dogs, horses and humans. Recent studies of the molecular pathology of a number of these startle syndromes have revealed that they are caused by defects in the inhibitory glycinergic pathways that mediate reciprocal and recurrent inhibition in the spinal cord. These defects arise from various mutations of the receptor for glycine, which either impair its sensitivity to agonists or reduce its expression in vivo. The emergent models of the molecular mechanisms that underlie startle disorders illustrate how diverse mutations can converge physiologically to produce a common phenotype.