title: Molecular analysis of the A322D mutation in the GABA receptor alpha-subunit causing juvenile myoclonic epilepsy.
abstract: 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.
authors: Neurologische Klinik, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. krampfl.klaus@mh-hannover.de
authors: Klaus
authors: K
authors: Krampfl
authors: Snezana
authors: S
authors: Maljevic
authors: Patrick
authors: P
authors: Cossette
authors: Elke
authors: E
authors: Ziegler
authors: Guy A
authors: GA
authors: Rouleau
authors: Holger
authors: H
authors: Lerche
authors: Johannes
authors: J
authors: Bufler
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D058785:GABA-A Receptor Agonists; D049452:Green Fluorescent Proteins; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D020190:Myoclonic Epilepsy, Juvenile / Q000235:genetics*; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
chemical_list: D058785:GABA-A Receptor Agonists; C546240:GABRA1 protein, human; D011963:Receptors, GABA-A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid