Johannes Bufler

A mutation in the GABAA receptor α1-subunit is associated with absence epilepsy

To detect mutations in GABRA1 in idiopathic generalized epilepsy.

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 ε-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.

Molecular analysis of the A322D mutation in the GABAA receptor α1‐subunit causing juvenile myoclonic epilepsy

Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy, the idiopathic generalized epilepsies. Although the mode of inheritance is usually complex, mutations in single genes have been shown to cause the disease in some families with autosomal dominant inheritance. The first mutation in a multigeneration JME family has been recently found in the α1‐subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing α1‐, β2‐ and γ2‐subunits in human embryonic kidney (HEK293) cells. By using an ultrafast application system, mutant receptors have shown reduced macroscopic current amplitudes at saturating GABA concentrations and a highly reduced affinity to GABA compared to the wild‐type (WT). Dose–response curves for current amplitudes, activation kinetics, and GABA‐dependent desensitization parameters showed a parallel shift towards 30‐ to 40‐fold higher GABA concentrations. Both deactivation and resensitization kinetics were considerably accelerated in mutant channels. In addition, mutant receptors labelled with enhanced green fluorescent protein (EGFP) were not integrated in the cell membrane, in contrast to WT receptors. Therefore, the A322D mutation leads to a severe loss‐of‐function of the human GABAA receptor by several mechanisms, including reduced surface expression, reduced GABA‐sensitivity, and accelerated deactivation. These molecular defects could decrease and shorten the resulting inhibitory postsynaptic currents (IPSCs) in vivo, which can induce a hyperexcitability of the postsynaptic membrane and explain the occurrence of epileptic seizures.

Control of kinetic properties of GluR2 flop AMPA‐type channels: impact of R/G nuclear editing

The GluR2 flop subunit of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐type glutamate receptors greatly determines calcium permeability and kinetic properties of heteromeric AMPA subunit assemblies. Post‐transcriptional editing of this subunit at the Q/R/N site controls calcium permeability whereas editing at the R/G site is involved in the regulation of biophysical properties. We used patch‐clamp techniques with ultrafast solution exchange to examine the kinetics of recombinant human homomeric GluR2 flop channels transiently expressed in HEK293 cells [edited at the R/G site and Q/R/N site (GR), and unedited (RN) and edited (GN) at the R/G site both with asparagine (N) at the Q/R/N site]. The time constant of desensitization after application of 10u2003mm glutamate was 1.38u2003±u20030.05u2003ms (nu2003=u200310), 5.53u2003±u20030.57u2003ms (nu2003=u20037) and 1.33u2003±u20030.06u2003ms (nu2003=u200312) for the GluR2 flop GR, RN and GN channels, respectively. The time constant of resensitization was 75u2003ms for the GluR2 flop RN and 30u2003ms for the GN channels. The dose‐dependence of the peak current amplitude, kinetics of activation and deactivation, and peak open probability did not differ between RN and GN channels. The study shows that desensitization and resensitization kinetics of homomeric GluR2 flop channels are controlled by a single amino acid exchange (glycine by arginine) at the R/G site. Quantitative analysis by computer simulation using a circular kinetic scheme allows the prediction of the main experimental results.

Kinetic properties of human AMPA‐type glutamate receptors expressed in HEK293 cells

AMPA‐type glutamate receptors (AMPAR) display a high variability in functional properties, which determine the time course of excitatory postsynaptic potentials. They are assembled as tetramers of GluR subunits 1–4 of different splice variants and nuclear edited isoforms. Presently, the kinetics of activation, desensitization and recovery from desensitization of human AMPARs (GluR1, 3 and 4 flip and flop, and GluR2 flip and flop in R and G edited forms, respectively) transiently expressed in HEK293 cells were studied with patch‐clamp techniques and ultra fast agonist application. Activation time constants were identical for all receptors (0.13u2003ms). The GluR2 flip G variant showed the slowest desensitization (10.8u2003ms), GluR4 flip the fastest (1.6u2003ms). Recovery from desensitization varied between 3.1u2003ms (GluR4 flip) and 178u2003ms (GluR1 flip). To determine functional interactions between subunits in heteromeric receptors the GluR1 flip and the GluR2 flip R were coexpressed. The time constant of desensitization increased linearly from 2.5u2003ms (GluR1 flip homomers) to 6.8u2003ms (GluR2 flip R homomers) with the amount of GluR2 flip R cDNA transfected. Recovery followed a monoexponential time course and had a time constant of 178u2003ms in GluR1 flip homomeric expression. In all GluR1 flip/GluR2 flip heteromers and in GluR2 flip R homomers desensitization recovered with a time constant of ≈50u2003ms. Thus, subunit interaction seems likely during recovery but not desensitization. Both parameters might influence the ability of AMPA receptors to mediate glutamate induced chronic excitotoxicity in neurodegenerative diseases.

Congenital myasthenic syndrome due to a novel missense mutation in the gene encoding choline acetyltransferase

Congenital myasthenic syndromes are caused by different genetic defects affecting proteins expressed at the neuromuscular junction. Recently, the first molecular genetic defect resulting in a presynaptic congenital myasthenic syndrome has been reported: Recessive loss-of-function mutations in CHAT, the gene encoding choline acetyltransferase, were described in five congenital myasthenic syndrome families. In this study, we investigated three patients from two independent Turkish kinships. Clinically, all patients presented with moderate myasthenic symptoms including ptosis and muscle weakness with increased fatigability. Multiple episodes of sudden apnea were reported for all patients. One child suffering from a second, unrelated disorder, i.e. hepatocellular carcinoma, showed a severe myasthenic phenotype, requiring permanent ventilation. Genetically, we identified a novel missense mutation (I336T) in the CHAT gene homozygously in all three patients. Haplotype analysis revealed that the mutant allele cosegregates with the clinical phenotype in both families (maximum combined two-point LOD-score of 2.46 for D10S1793). In summary, we confirm that CHAT mutations are responsible for a clinically distinct form of congenital myasthenic syndrome, characterized by episodic apnea. Infections and stress may lead to a life-threatening failure of neuromuscular transmission in congenital myasthenic syndrome with episodic apnea. The observation of the same mutation (I336T) in two independent Turkish kinships may suggest a common origin, i.e. founder.

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. Muscle Nerve 27:435–441, 2003

Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons

AMPA-type glutamate receptor (GluR) channels provide fast excitatory synaptic transmission in the CNS, but mediate also cytotoxic insults. It could be shown that AMPA-type GluR channel-mediated chronic excitotoxicity leads to an increased intracellular calcium concentration and plays an important role in neurodegenerative diseases like for example amyotrophic lateral sclerosis (ALS). As calcium is an important mediator of various processes in the cell and calcium signals have to be very precise in the temporospatial resolution, excessive intracellular calcium increases can seriously impair cell function. It is still unclear if AMPA-type receptors can directly interact with the intracellular calcium homeostasis or if other mechanisms are involved in this process. The objective of this study was therefore to investigate the calcium homeostasis in rat motoneurons under physiological stimulation of AMPA-type GluR channels using calcium imaging techniques and patch-clamp recordings simultaneously. It was found that spontaneous excitatory postsynaptic currents of cultured motoneurons did not elicit significant intracellular calcium transients. Large intracellular calcium transients occurred only when preceding fast sodium currents were observed. Pharmacological experiments showed that activation of AMPA-type GluR channels during synaptic transmission has a great functional impact on the calcium homeostasis in motoneurons as all kinds of activity was completely blocked by application of the selective kainate- and AMPA-type GluR channel blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Furthermore we suggest from our experiments that calcium transients of several hundred milliseconds duration result from release of calcium from the endoplasmic reticulum via activation of ryanodine receptors (calcium-induced calcium release, CICR). Our results help to understand the regulatory function of AMPA-type GluR channels in the intracellular calcium homeostasis which is known to be disturbed in neurodegenerative diseases.

Desensitization characteristics of rat recombinant GABAA receptors consisting of α1β2γ2S and α1β2 subunits expressed in HEK293 cells

Abstract Desensitization kinetics of rat recombinant typeA GABAergic receptors consisting of the subunits α1β2γ2S or α1β2 was investigated on application of 10–0.001 mM GABA to whole cell patches using a piezo driven liquid filament switch for fast application and deapplication. At high GABA concentrations desensitization was triphasic showing increasing time constants and a decreasing extent of desensitization on lowering the GABA concentration. Below agonist concentrations of 1 mM for the trimeric receptor and 0.1 mM for the dimeric one desensitization was biphasic switching to monophasic kinetics at GABA concentrations ≤0.01 mM for the α1β2γ2S-type and ≤0.003 mM for the α1β2-type, respectively. Comparison with former studies performed with GABAergic receptors consisting of different subunits revealed differences in the desensitization kinetics.

Competitive and open channel block of recombinant nAChR channels by different antibiotics

Various antibiotics may impair neuromuscular transmission, provoking symptoms of myasthenia in patients with a compromised safety margin of the synaptic transmission, but little is known about the underlying mechanisms at the molecular level. Using a modified patch-clamp technique in combination with an ultrafast system for solution exchange we investigated the functional interaction of gentamicin, penicillin G, tetracycline, erythromycin and ceftriaxone with nAChR transiently transfected into HEK293 cells as a potential molecular target. Gentamicin, penicillin G, tetracycline and erythromycin induced a combination of open channel and competitive block of nAChR channel currents whereas ceftriaxone had no effect. The IC50 for the competitive block was close to or within the range of clinically relevant concentrations. Except for erythromycin the open channel block was observed only at higher concentrations. From our in-vitro results we conclude that competitive inhibition of nAChR channels by antibiotics is an important mechanism underlying the impairment of neuromuscular transmission under clinical conditions.