Prevalence and prognostic impact of comorbidities in amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is characterized by rapidly progressive paralysis of striated muscles due to the loss of upper and lower motor neurons. The disease leads to death within 2-5 years, mainly due to respiratory failure. The pathogenesis of ALS is still unexplained for the most part. In this study, we aimed to determine the prevalence of different cardiovascular, metabolic, and neuropsychiatric comorbidities in a large ALS cohort and to evaluate their influence on the disease course. METHODS: A cohort of 514 patients with ALS of our ALS outpatient clinic was investigated retrospectively with reference to known prognostic factors and comorbidities. The prevalence of concomitant diseases was compared with the data from the German general population. Uni- and multivariate survival analyses were performed using the Cox proportional hazards model and Kaplan-Meier analysis. RESULTS: The prevalence of cardiovascular diseases and cardiovascular risk factors was significantly lower in patients with ALS compared to the German general population, whilst the prevalence of dementia, parkinsonism, and depressive symptoms was significantly higher in the ALS cohort. None of the investigated comorbidities had an influence on the disease course or on the survival of patients. CONCLUSIONS: Persons with cardiovascular diseases or risk factors seem to be at lower risk of ALS. Although these diseases are apparently somehow protective regarding ALS susceptibility, their presence did not modify disease progression and survival in patients with ALS. Our study further confirms the well-known continuum between ALS and dementia. It also suggests a link with other neurodegenerative diseases such as Parkinson's disease.

Modulation of synaptic transmission and analysis of neuroprotective effects of valproic Acid and derivates in rat embryonic motoneurons.

Amyotrophic lateral sclerosis is a devastating motoneuron disorder for which no effective treatment exists. There is some evidence for neuroprotective effects of valproic acid (VPA). The beneficial effects, however, are limited due to the adverse effects of VPA. To overcome this problem, a number of VPA derivates with fewer side effects have been synthesized. In the present study, we investigated the viability of highly purified embryonic motoneurons cultured on glial feeder layers, composed of either astrocytes or Schwann cells, or in monoculture, in presence of VPA and its three derivates 3-propyl-heptanoic acid (3-PHA), PE-4-yn enantiomers (R- and S-PE-4-yn). An excitotoxic stimulus, kainate (KA), was added at day in vitro 9 (DIV9) and the neuroprotective effect of either simultaneous incubation (DIV9) or pre-incubation (DIV1) of VPA and its derivates was tested. The survival of motoneurons under simultaneous application of KA and VPA derivates was not remarkably increased. Pre-incubation with VPA and even more with the derivates before the addition of KA, however, significantly reduced their vulnerability against the KA-induced neurotoxic effect. Our data suggest that the neuroprotective capacities of VPA and its three derivates tested here drastically increase when they are added several days before KA. Most prominent neuroprotective effects were seen for the PE-4-yn enantiomers. Patch-clamp experiments revealed an antiexcitotoxic effect of the S-PE-4-yn enantiomer that reduces the frequency of postsynaptic currents and enhances the inhibitory postsynaptic transmission dependent on the co-culture condition.

Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study.

Gamma-aminobutyric acid receptor type A (GABA(A)) receptor channels mediate fast inhibitory neurotransmission throughout the central nervous system while the expression of ionotropic glycine receptors is mainly restricted to the spinal cord and brain stem. Neuroactive steroids are well known as positive allosteric modulators of GABA(A) receptor function. Furthermore, there have been hints for an interaction of neuroactive steroids with ionotropic glycine receptors. The aim of the study was to characterize the effect of androsterone and progesterone on alpha(1) and alpha(1)beta glycine receptor and alpha(1)beta(2)gamma(2) GABA(A) receptor channels and to examine the molecular interactions between ligands and receptors. Electrophysiological recordings were performed on HEK 293 cells using the patch clamp technique in combination with an ultrafast perfusion system. A direct activation of inhibitory ionotropic receptors was observed for androsterone at GABA(A) receptor channels. A coactivation of currents elicited by nonsaturating agonist concentrations was observed with androsterone and progesterone at glycine and GABA(A) receptor channels. We could show that association of beta subunits with alpha subunits affects the sensitivity of glycine receptors to androsterone. In contrast to previous reports in which recombinant glycine receptors were inhibited by progesterone, a potentiating effect was revealed by our experiments. At concentrations of 0.1 mM and higher, there were also hints to a channel block-like mechanism. In conclusion, different molecular mechanisms of interaction between neuroactive steroids and GABA as well as glycine receptors could be identified and quantitatively described. Our data clarify the role of steroid compounds in the modulation of inhibitory receptor channel function.

Analysis of neuroprotective effects of valproic acid on primary motor neurons in monoculture or co-cultures with astrocytes or Schwann cells.

Chronic dysregulation of the intracellular Ca(2+) homeostasis (excitotoxicity) is thought to contribute to the development of motor neuron diseases. Valproic acid (VPA) is widely used as an antiepileptic drug and acts mainly by inhibition of sodium channels and by enhancing the level of the inhibitory neurotransmitter gamma-aminobutyric acid. Neuroprotective capacities of VPA are supposed to arise also from the inhibition of histone deacetylases. We investigated the viability of highly purified rat embryonic motor neurons cultured on glial feeder layers, composed of either astrocytes or Schwann cells, or in the absence of glia, monoculture in presence of VPA and/or kainate (KA) using immunocytochemistry and calcium imaging. A significant effect of the culture and co-culture conditions on the viability of motor neurons in our in vitro model of excitotoxicity was detected. The neuroprotective effect of VPA on primary embryonic motor neuron cultures was not proven. A functional interaction between VPA and KA occurred during the first 10 days in culture.

A long-term follow-up of botulinum toxin A in cervical dystonia.

OBJECTIVE: Cervical dystonia (CD) is the most common form of adult-onset focal dystonia, and botulinum toxin A (BoNT-A) has become the first-line treatment for this condition. METHODS: In this work, we present data of 207 CD patients treated with BoNT-A for 6.7 +/- 3.5 years. One hundred and sixty-three patients were treated with Dysport (mean dose, 389 +/- 144 U) and 44 with Botox (mean dose, 145 +/- 44 U). RESULTS: The mean clinical benefit, based on a 0-3 scale (0=no effect, 1=slight, 2=moderate and 3=marked improvement) was similar for Dysport (2.5 +/- 0.3) and Botox (2.2 +/- 0.4). Adverse events were mild and similar for both products. Fewer than 2% of the patients developed neutralizing antibodies. DISCUSSION: These data confirm the efficacy and safety of BoNT-A treatment in CD over an extended period of up to 14 years.

Anterior cysts of the spine: a difficult differential diagnosis to amyotrophic lateral sclerosis.

We describe three patients referred to our ALS/MND clinic with suspected diagnosis of amyotrophic lateral sclerosis (ALS). The patients were all male, middle aged, and their initial symptoms were weakness and fasciculations in upper limb muscles. Results of clinical and electrophysiological examination in all cases were in accordance with possible ALS according to the revised El Escorial criteria. Other conditions mimicking ALS appeared to be excluded by extensive technical examinations and laboratory tests. Only repeated MRI examinations revealed anterior spinal cysts several years after symptom onset. This report intends to highlight this rare and difficult differential diagnosis of ALS and underlines the value of the revised El Escorial criteria in conjunction with electrophysiology to asses the certainty of the diagnosis ALS.

Molecular mechanisms of interaction between the neuroprotective substance riluzole and GABA(A)-receptors.

The antiepileptic drug riluzole is used as a therapeutic agent in amyotrophic lateral sclerosis due to its neuroprotective effects. Besides presynaptic inhibition of GABAergic and preferentially glutamatergic transmission, it also potentiates postsynaptic GABA(A)-receptor function. We investigated the postsynaptic effects of riluzole on GABA(A)-receptor channels by use of the patch-clamp technique. Recombinant alpha1beta2gamma(2s) and alpha1beta2 GABA(A) receptors were expressed in HEK 293 cells by transient transfection. Pulses of GABA were applied in combination with different concentrations of riluzole to whole cell or outside-out patches with either alpha1beta2gamma(2s) or alpha1beta2 GABA(A)-receptor channels. Co-application of riluzole led to a slight decrease of absolute peak current amplitudes and steady-state currents in prolonged presence of GABA at saturating concentrations. In the presence of riluzole, enhancement of current amplitudes was observed with lower concentrations of GABA at alpha1beta2gamma(2s) receptors and to a lower extent also at alpha1beta2 receptors. Thus, the potentiating effect of riluzole was shown to be not abolished in the absence of the gamma(2s)-subunit. A further prominent effect of riluzole was a highly significant acceleration of the time course of current decay, most probably pointing to an open-channel block-like mechanism of action. As both receptor subtypes were affected similarly by the block, it could be concluded that the respective binding sites should be assumed within a region of high sequence homology like it is given for the channel-lining M2 domain of GABA(A)-receptor subunits. In conclusion, three different molecular mechanisms of interaction of the neuroprotective compound riluzole were observed at two different subtypes of GABA(A) receptor channels. The results further point to the impact of the inhibitory as well as the excitatory synaptic activity as a pharmacological target to counteract chronic excitotoxicity and reveal molecular mechanisms of action of the only one neuroprotective drug in current clinical use in patients suffering from amyotrophic lateral sclerosis.

The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice.

ALS is a fatal neurodegenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. About 10% of ALS cases are familial, in 10-20% of these, mutations in the gene coding for superoxide dismutase 1 (SOD1) can be detected. Overexpression of mutated SOD1 in mice created animal models which clinically resemble ALS. Abnormalities in glutamatergic and GABAergic neurotransmission presumably contribute to the selective motor neuron damage in ALS. By in situ hybridization histochemistry (ISH), we investigated the spinal mRNA expression of the GABAA and AMPA type glutamate receptor subunits at different disease stages on spinal cord sections of mutant SOD1 mice and control animals overexpressing wild-type SOD1 aged 40, 80, 120 days and at disease end-stage, i.e. around 140 days) (n=5, respectively). We detected a slight but statistically significant decrease of the AMPA receptor subunits GluR3 and GluR4 only in end stage disease animals.

High dose vitamin E therapy in amyotrophic lateral sclerosis as add-on therapy to riluzole: results of a placebo-controlled double-blind study.

UNLABELLED: Increasing evidence has suggested that oxidative stress may be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). The antioxidant vitamin E (alpha-tocopherol) has been shown to slow down the onset and progression of the paralysis in transgenic mice expressing a mutation in the superoxide dismutase gene found in certain forms of familial ALS. The current study, a double blind, placebo-controlled, randomised, stratified, parallel-group clinical trial, was designed to determine whether vitamin E (5000 mg per day) may be efficacious in slowing down disease progression when added to riluzole. METHODS: 160 patients in 6 German centres with either probable or definite ALS (according to the El Escorial Criteria) and a disease duration of less than 5 years, treated with riluzole, were included in this study and were randomly assigned to receive either alpha-tocopherol (5000 mg per day) or placebo for 18 months. The Primary outcome measure was survival, calculating time to death, tracheostomy or permanent assisted ventilation, according to the WFN-Criteria of clinical trials. Secondary outcome measures were the rate of deterioration of function assessed by the modified Norris limb and bulbar scales, manual muscle testing (BMRC), spasticity scale, ventilatory function and the Sickness Impact Profile (SIP ALS/19). Patients were assessed at entry and every 4 months thereafter during the study period until month 16 and at a final visit at month 18. Vitamin E samples were taken for compliance check and Quality Control of the trial. For Safety, a physical examination was performed at baseline and then every visit until the treatment discontinuation at month 18. Height and weight were recorded at baseline and weight alone at the follow-up visits. A neurological examination as well as vital signs (heart rate and blood pressure), an ECG and VEP's were recorded at each visit. Furthermore, spontaneously reported adverse experiences and serious adverse events were documented and standard laboratory tests including liver function tests performed. For Statistical Analysis, the population to be considered for the primary outcome measure was an "intent-to-treat" (ITT) population which included all randomised patients who had received at least one treatment dose (n = 160 patients). For the secondary outcome measures, a two way analysis of variance was performed on a patient population that included all randomised patients who had at least one assessment after inclusion. RESULTS: Concerning the primary endpoint, no significant difference between placebo and treatment group could be detected either with the stratified Logrank or the Wilcoxon test. The functional assessments showed a marginal trend in favour of vitamin E, without reaching significance. CONCLUSION: Neither the primary nor the secondary outcome measures could determine whether a megadose of vitamin E is efficacious in slowing disease progression in ALS as an add-on therapy to riluzol. Larger or longer studies might be needed. However, administration of this megadose does not seem to have any significant side effects in this patient population.

Ligand-gated channels in early mesencephalic neuronal precursors: immunocytochemical and electrophysiological analysis.

Neuronal precursors play an important role in potential regenerative therapeutic strategies in different neurodegenerative diseases, e.g. Parkinson's disease. To understand proliferation and differentiation of these cells in vitro and in vivo, it is important to characterize functional properties of neuronal precursors in detail. The aim of the present study was to analyse the electrophysiological characteristics of ligand-gated channels of neuronal precursors prepared from the rat ventral mesencephalon (VM) of embryonic stage 12.5 during their in vitro differentiation. For the experiments we used the patch-clamp technique in combination with a system for ultrafast solution exchange and immunocytochemistry. It could be shown that functional active AMPA-type glutamate as well as GABA(A) receptor channels are expressed at an early stage of neuronal development. In culture we observed excitatory as well as inhibitory postsynaptic currents (defined by their different kinetics) which correspond to the activation of AMPAergic and GABAergic receptor channels. Two populations of glutamate-activated currents could be differentiated by their different time course of desensitization whereas the time course of resensitization and deactivation was normally distributed in all cells. GABAergic currents could be blocked by bicuculline and their kinetics correspond to that of GABA(A) receptor channel currents. Summarizing the results, in the present study it was shown for the first time that neuronal embryonic precursors of the rat VM express both functional AMPA-type glutamate and functional GABA(A) receptor channels in vitro.

Kinetic analysis of recombinant mammalian alpha(1) and alpha(1)beta glycine receptor channels.

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.

Rapsyn N88K is a frequent cause of congenital myasthenic syndromes in European patients.

BACKGROUND: Mutations in various genes of the neuromuscular junction may cause congenital myasthenic syndromes (CMS). Most mutations identified to date affect the epsilon-subunit gene of the acetylcholine receptor (AChR), leading to end-plate AChR deficiency. Recently, three different mutations in the RAPSN gene have been identified in four CMS patients with AChR deficiency. OBJECTIVE: To perform mutation analysis of the RAPSN gene in patients with sporadic or autosomal recessive CMS. METHODS: One hundred twenty CMS patients from 110 unrelated families were analyzed for the RAPSN mutation N88K by restriction fragment length polymorphism and sequence analysis. RESULTS: In 12 CMS patients from 10 independent families, RAPSN N88K was identified either homozygous or heteroallelic to another missense mutation. Symptoms usually started perinatally or in the first years of life. However, one patient did not show any myasthenic symptoms before the third decade. Clinical symptoms typically included bilateral ptosis, weakness of facial, bulbar, and limb muscles, and a favorable response to anticholinesterase treatment. Crisis-like exacerbations with respiratory insufficiency provoked by stress, fever, or infections in early childhood were frequent. All RAPSN N88K families originate from Central or Western European countries. Genotype analysis indicated that they derive from a common ancestor (founder). CONCLUSIONS: The RAPSN mutation N88K is a frequent cause of rapsyn-related CMS in European patients. In general, patients (RAPSN N88K) were characterized by mild to moderate myasthenic symptoms with favorable response to anticholinesterase treatment. However, severity and onset of symptoms may vary to a great extent.

IgG from patients with Guillain-Barré syndrome interact with nicotinic acetylcholine receptor channels.

In Guillain-Barré syndrome (GBS), immunoglobulin G (IgG) antibodies block neuromuscular transmission pre- and postsynaptically and thus are of potential pathogenic relevance. We investigated whether IgG from GBS patients has a direct interaction with nicotinic acetylcholine receptor (nAChR) channels. Purified IgG fractions from six GBS patients that blocked neuromuscular transmission in a previous study were analyzed by the patch-clamp technique in combination with an ultrafast system for solution exchange. Sera from three patients with other inflammatory neurological disorders were used as controls. Mouse myotubes expressing native embryonic-type nAChR channels and human embryonic kidney (HEK) 293 cells transiently transfected with recombinant adult-type nAChR channels were used. Repeated 20-ms pulses of acetylcholine (ACh) were applied to outside-out patches in the presence of GBS-IgG. IgG of the patients had a significant reversible blocking action on embryonic- and adult-type nAChR channels with some variability in the magnitude of the block. Activation and desensitization kinetics were not affected when GBS-IgG was applied. None of the control sera blocked the AChR channels. The observed postsynaptic block effect fulfills the criteria of a channel-blocking IgG antibody similar to those seen in autoimmune myasthenia and may contribute to muscle weakness during the acute phase of GBS.

[Cerebellar symptoms in motor neuron diseases. Special form of amyotrophic lateral sclerosis plus syndrome]. / Zerebelläre Symptome bei Motoneuronerkrankungen. Spezielle Form des ALS-Plus-Syndroms.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive and selective loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. The upper motor neuron syndrome is characterized by symptoms of spastic paresis. Muscle weakness and atrophy, fasciculations, and cramps are typical signs for the degeneration of the lower motor neurons. In 1994, the El Escorial criteria were proposed for the diagnosis of ALS. These criteria include ALS-plus syndromes, which are defined by an association of ALS with extrapyramidal features or dementia. In this paper, we present two cases of ALS associated with signs of cerebellar degeneration. According to the revised El Escorial criteria, the described unusual combination of upper and lower motor neuron signs in association with cerebellar ataxia can be classified as a specific form of ALS-plus syndromes.

Analysis of a slow desensitized state of recombinant adult-type nicotinic acetylcholine receptor channels.

A characteristic feature of the kinetics of nicotinic acetylcholine receptor (nAChR) channels is fast and nearly complete desensitization with a time course between 10 and 100 ms and recovery from desensitization in the range of some hundred ms. In the present study we used a piezo-driven system for ultra-fast solution exchange, analysed the recovery from the fast desensitized state of mouse recombinant adult-type nAChR channels and found no difference to that of embryonic-type channels. By double pulse experiments with application of pulses with a saturating concentration of 1 mm acetylcholine (ACh) with increasing duration of the first pulse and a constant interval between pulses we detected a second slow desensitized state which was entered with a time constant of 2835 ms. Recovery from the slow desensitized state proceeded with a single exponential with a time constant of 16134 ms. The experimental data were interpreted by the addition of a transition from the desensitized state with two bound ACh molecules to a slow desensitized state to the well known circular kinetic scheme of activation and desensitization of nAChR channels. This slow desensitized state might play a role in muscle fatigue or in pathological states like myasthenic syndromes.

Pentobarbital and brilliant green modulate the current response of recombinant rat kainate-type GluR6 receptor channels differentially.

Kainate-type receptor channels (GluR5-7, KA1,2) belong to the family of ionotropic glutamate receptor channels. In the present study we tested the interaction of two different drugs with GluR6 channels using outside-out patches from HEK cells transiently transfected with cDNA of GluR6 channels. Glutamate and the respective drugs were delivered by a system for ultrafast solution exchange. Application of a saturating concentration of 3 mM glutamate resulted in fast current transients with desensitization time constants between 3 and 10 ms. Addition of pentobarbital (>or=1 mM) to the 3 mM glutamate containing test-solution resulted in a significant decrease of the time constant of current decay without affecting the peak current amplitude. Brilliant green (>or=1 mM) had the opposite effect and led to an increase of the time constant of current decay after application of 3 mM glutamate. The pharmacological effects of both drugs were completely reversible. Additionally, a significant increase of the peak current amplitude and the time constant of deactivation in presence of brilliant green was observed. Summarizing our results, we could identify a further substance, brilliant green, interacting with GluR6 kainate-type receptor channels.

Functional diversity of recombinant human AMPA type glutamate receptors: possible implications for selective vulnerability of motor neurons.

Lower motor neurons are known to be susceptible to glutamate-mediated cell damage via overstimulation of AMPA type glutamate receptors (GluR). The molecular basis of an important hypothesis in investigating amyotrophic lateral sclerosis (ALS) is glutamate-excitotoxicity. The aim of this study was to define desensitization and deactivation kinetics of recombinant human GluR1 and GluR2 receptor channels and their splice variants by means of patch-clamp experiments employing ultrafast solution exchange techniques. By this approach, the desensitization time constants of homooligomeric channels could be measured as tau(Des)=2.95+/-0.22 ms (n=10) for GluR1flip, tau(Des)=3.17+/-0.19 ms (n=10) for GluR1flop, tau(Des)=9.86+/-0.79 ms (n=10) for GluR2flip, and tau(Des)=1.87+/-0.26 ms (n=10) for GluR2flop, respectively. In the case of GluR1flip/flop and GluR2flop, a nondesensitising steady state current of less than 1% of peak current amplitude was observed, while GluR2flip channel currents showed a marked steady state component of about 10% of the maximum current. No significant differences were detected comparing the deactivation time course of GluR1 and GluR2 splice variants. These results suggest that the human GluR subtypes tested comprise no fundamental difference to their rodent analogous. Therefore, we describe a preparation that will be useful for further investigation of motor neuron physiological properties and a methodological approach allowing to study functional recombinant human GluR channels under reliable conditions.

Structural requirements of phenol derivatives for direct activation of chloride currents via GABA(A) receptors.

Propofol directly activates gamma-aminobutyric acid (GABA(A)) receptors in the absence of the natural agonist. This mechanism is supposed to contribute to its sedative-hypnotic actions. We studied the effects of seven structurally related phenol derivatives on chloride inward currents via rat alpha1beta2gamma2 GABA(A) receptors, heterologously expressed in HEK 293 cells in order to find structural determinants for this direct agonistic action. Only compounds with the phenolic hydroxyl attached directly to the benzene ring and with aliphatic substituents in ortho position to the phenolic hydroxyl activated chloride currents in the absence of GABA. Concentrations required for half-maximum effect were 980 microM for 2-methylphenol, 230 microM for 2,6-dimethylphenol, 200 microM for thymol, and 23 microM for propofol. Drug-induced chloride currents showed no desensitisation during the 2-s application. These results show that the position of the aliphatic substituents with respect to the phenolic hydroxyl group is the crucial structural feature for direct GABA(A) activation by phenol derivatives.

Deactivation and desensitization of mouse embryonic- and adult-type nicotinic receptor channel currents.

Recombinant nicotinic acetylcholine receptor (nAChR) channels transiently expressed in HEK293 cells were investigated using the patch-clamp technique in the cell-attached and outside-out modes for single-channel analysis and ultra-fast agonist application to multiple channels. Deactivation (current decay after removal of agonist) and desensitization (current decay in the presence of agonist) were analyzed at embryonic- (gamma) and adult-type (epsilon) nAChR channels. Time constants of desensitization were similar for both receptor types (epsilon: 53.1+/-16.9 ms; gamma: 49.2+/-15.7 ms) and corresponded to the mean duration of clusters of single channel openings activated by pulses of 1 mM ACh. Deactivation showed distinct characteristics. Time constants were 1.76+/-0.16 ms for epsilon- and 3.19+/-0.18 ms for gamma-nAChR channels, corresponding to mean burst duration analyzed from single channels in the same preparation (epsilon: 1.85+/-1.2 ms, gamma: 3.85+/-2.1 ms). It is assumed that differences in deactivation are of functional relevance at the muscle endplate.

Interaction of the neuroprotective drug riluzole with GABA(A) and glycine receptor channels.

Riluzole is used as therapeutic agent in amyotrophic lateral sclerosis. We investigated the interaction of riluzole with recombinant GABA (gamma-aminobutyric acid)(A) receptor channels (alpha(1)beta(2)gamma(2)-subunits) and glycine receptor channels (alpha(1)beta-subunits) transiently expressed in HEK293 cells. For electrophysiological experiments, the patch-clamp technique in combination with tools for ultrafast solution exchange was used. Saturating concentrations of GABA or glycine were applied with different concentrations of riluzole to outside-out patches containing alpha(1)beta(2)gamma(2) GABA(A) receptor channels or alpha(1)beta-glycine receptor channels on their surface, respectively. The current declined after application of GABA or glycine with three time constants of desensitization to a steady-state current amplitude. Application of riluzole resulted in a shift to fast desensitized states at both receptors. The proportion of the time constants of fast desensitization increased and the time constants of slow desensitization and the steady-state current decreased whereas the maximal current amplitudes were not affected by riluzole. The data of the study demonstrate for the first time interaction of GABAergic and glycinergic currents with riluzole under physiological conditions.