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

Neurologische Klinik, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
European Journal of Neuroscience (Impact Factor: 3.18). 08/2005; 22(1):10-20. DOI: 10.1111/j.1460-9568.2005.04168.x
Source: PubMed


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 alpha1-subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing alpha1-, beta2- and gamma2-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.

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    • "To explore both of these issues, we examined the subcellular distribution of mutant a1 subunits that had been co-expressed with wildtype b2 and c2 subunits (Figs 3 and 4). As controls, we compared the staining pattern of the wildtype a1 subunit which traffics normally and the a1 A322D mutant which accumulates in the ER and exhibits reduced plasma-membrane surface expression (Cossette et al., 2002; Krampfl et al., 2005; Maljevic et al., 2006). As antibody recognition for the myc-tagged c2 GABA A receptor subunit was variable, we focused only on examining its functional properties using electrophysiology (see below). "
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    ABSTRACT: Epilepsy is a heterogeneous neurological disease affecting approximately 50 million people worldwide. Genetic factors play an important role in both the onset and severity of the condition, with mutations in several ion-channel genes being implicated, including those encoding the GABA(A) receptor. Here, we evaluated the frequency of additional mutations in the GABA(A) receptor by direct sequencing of the complete open reading frame of the GABRA1 and GABRG2 genes from a cohort of French Canadian families with idiopathic generalized epilepsy (IGE). Using this approach, we have identified three novel mutations that were absent in over 400 control chromosomes. In GABRA1, two mutations were found, with the first being a 25-bp insertion that was associated with intron retention (i.e. K353delins18X) and the second corresponding to a single point mutation that replaced the aspartate 219 residue with an asparagine (i.e. D219N). Electrophysiological analysis revealed that K353delins18X and D219N altered GABA(A) receptor function by reducing the total surface expression of mature protein and/or by curtailing neurotransmitter effectiveness. Both defects would be expected to have a detrimental effect on inhibitory control of neuronal circuits. In contrast, the single point mutation identified in the GABRG2 gene, namely P83S, was indistinguishable from the wildtype subunit in terms of surface expression and functionality. This finding was all the more intriguing as the mutation exhibited a high degree of penetrance in three generations of one French Canadian family. Further experimentation will be required to understand how this mutation contributes to the occurrence of IGE in these individuals.
    European Journal of Neuroscience 06/2011; 34(2):237-49. DOI:10.1111/j.1460-9568.2011.07767.x · 3.18 Impact Factor
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    • "MEG and beamformer analysis using simple motor paradigms therefore offers an attractive framework to study disease as well as healthy (Jurkiewicz et al., 2006; Cheyne et al., 2008) and developmental brain function (Gaetz et al., 2010). Further , given the link between cortical oscillations and GABAergic mechanisms in visual cortex (Muthukumaraswamy et al., 2009a) and sources that suggest altered GABAergic dysfunction in JME, ranging from genetic (Noebels, 2002; Krampfl et al., 2005; Ma et al., 2006; Macdonald et al., 2010), in vitro (Rainesalo et al., 2003) and TMS studies (Badawy et al., 2010) we postulated that there would be differences in induced cortical responses in patients with JME compared to controls during simple visual and motor tasks. We chose to study the motor and visual systems in JME, comparing cortical responses in a patient and control group using simple visual and motor paradigms known to produce robust induced responses in cortical oscillatory rhythms. "
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    ABSTRACT: We investigated differences in task induced responses in occipital and sensorimotor cortex between patients with juvenile myclonic epilepsy (JME) and healthy controls . Twelve patients with JME and 12 age-matched non-epilepsy volunteers performed visual and motor tasks during MEG. We used synthetic aperture magnetometry to localise areas of task-related oscillatory modulations, performed time-frequency analyses on the locations of peak task related power changes and compared power and frequency modulation at these locations between patients and controls. Patients with JME had significantly reduced pre-movement beta event-related desynchronisation in the motor task compared to controls. No significant differences were seen in other motor-related responses, or visual oscillatory responses. Altered beta event-related desynchronisation may represent network specific dysfunction in JME possibly through GABAergic dysfunction. Characterising task specific cortical responses in epilepsy offers the potential to understand the patho-physiological basis of seizures and provide a window on disease and treatment effects.
    Clinical neurophysiology: official journal of the International Federation of Clinical Neurophysiology 05/2011; 122(11):2128-38. DOI:10.1016/j.clinph.2011.04.017 · 3.10 Impact Factor
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    • "In addition, recent studies have identified the α 1 (A294D) and α 1 (A322D) genetic mutations of the GABA A R α 1 subunit that are associated with juvenile myoclonic epilepsy (Cossette et al., 2002; Gallagher et al., 2004; 2005; 2007; Mizielinska et al., 2006). These mutations affect GABA A R gating, expression, and/or trafficking of the receptor to the cell surface -all pathophysiological mechanisms that result in neuronal disinhibition, cause hyperexcitability throughout the brain, and predispose to epileptic seizures (Fisher, 2004; Krampfl et al., 2005; Macdonald et al., 2006). The mechanism by which the α 1 (A322D) mutation reduced total and surface α 1 (A322D) protein expression could be inhibition of correct GABA A R folding and assembly and/or alteration of channel gating properties (Gallagher et al., 2005). "
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    ABSTRACT: Photothrombotic infarcts of the neocortex result in structural and functional alterations of cortical networks, including decreased GABAergic inhibition, and can generate epileptic seizures within 1 month of lesioning. In our study, we assessed the involvement and potential changes of cortical GABA A receptor (GABA AR) alpha1 subunits at 1, 3, 7, and 30 days after photothrombosis. Quantitative competitive reverse transcription-polymerase chain reaction (cRT-PCR) and semi-quantitative Western blot analysis were used to investigate GABA AR alpha1 subunit mRNA and protein levels in proximal and distal regions of perilesional cortex and in homotopic areas of young adult Sprague-Dawley rats. GABA AR alpha1 subunit mRNA levels were decreased ipsilateral and contralateral to the infarct at 7 days, but were increased bilaterally at 30 days. GABA AR alpha1 subunit protein levels revealed no significant change in neocortical areas of both hemispheres of lesioned animals compared with protein levels of sham-operated controls at 1, 3, 7, and 30 days. At 30 days, GABA AR alpha1 subunit protein expression was significantly increased in lesioned animals within proximal and distal regions of perilesional cortex compared with distal neocortical areas contralaterally (Student's t-test, p<0.05). Short- and long-term alterations of mRNA and protein levels of the GABA AR alpha1 subunit ipsilateral and contralateral to the lesion may influence alterations in cell surface receptor subtype expression and GABA AR function following ischemic infarction and may be associated with formative mechanisms of poststroke epileptogenesis.
    Brain Research 06/2008; 1210:29-38. DOI:10.1016/j.brainres.2008.02.070 · 2.84 Impact Factor
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