Martin J. Gallagher

Mutations in GABAA receptor subunits associated with genetic epilepsies

Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking.

The GABAA receptor α1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation

A form of autosomal dominant juvenile myoclonic epilepsy is caused by a nonconservative missense mutation, A322D, in the GABAA receptor α1 subunit M3 transmembrane helix. We reported previously that the A322D mutation reduced total and surface α1(A322D) subunit protein and that residual α1(A322D) subunit resided in the endoplasmic reticulum. Here, we demonstrate that the reduction in α1(A322D) expression results from rapid endoplasmic reticulum-associated degradation of the α1(A322D) subunit through the ubiquitin–proteasome system. We provide direct evidence that the α1(A322D) subunit misfolds and show that in at least 33% of α1(A322D) subunits, M3 failed to insert into the lipid bilayer. We constructed a series of mutations in the M3 domain and empirically determined the apparent free energy cost (ΔGapp) of membrane insertion failure, and we show that the ΔGapp correlated directly with the recently elucidated transmembrane sequence code (ΔGLep). These data provide a biochemical mechanism for the pathogenesis of this epilepsy mutation and demonstrate that ΔGLep predicts the efficiency of lipid partitioning of a naturally occurring proteins transmembrane domain expressed in vivo. Finally, we calculated the ΔΔGLep for 277 known transmembrane missense mutations associated with Charcot–Marie–Tooth disease, diabetes insipidus, retinitis pigmentosa, cystic fibrosis, and severe myoclonic epilepsy of infancy and showed that the majority of these mutations also are likely to destabilize transmembrane domain membrane insertion, but that only a minority of the mutations would be predicted to be as destabilizing as the A322D mutation.

Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit

A GABAA receptor α1 subunit epilepsy mutation (α1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the α1 subunit. We reported previously that heterologous expression of α1(A322D)β2γ2 receptors in mammalian cells resulted in reduced total and surface α1 subunit protein. Here we demonstrate the mechanism of this reduction. Total α1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 ± 6%) or when coexpressed with β2 and γ2S subunits (78 ± 6%), indicating an expression reduction prior to subunit oligomerization. In α1β2γ2S receptors, endoglycosidase H deglycosylated only 26 ± 5% of α1 subunits, consistent with substantial protein maturation, but in α1(A322D)β2γ2S receptors, endoglycosidase H deglycosylated 91 ± 4% of α1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the α1 subunit was tagged with yellow (α1-YFP) or cyan (α1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 ± 4% of α1-YFPβ2γ2 but only 5 ± 1% α1(A322D)-YFPβ2γ2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 ± 4% α1-YFPβ2γ2S, 86 ± 3% α1(A322D)-YFP). Heterozygous expression of α1-CFPβ2γ2S and α1(A322D)-YFPβ2γ2S or α1-YFPβ2γ2S and α1(A322D)-CFPβ2γ2S receptors showed that membrane GABAA receptors contained primarily wild type α1 subunits. These data demonstrate that the A322D mutation reduces α1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane α1 subunits that are almost exclusively wild type subunits.

The Juvenile Myoclonic Epilepsy GABAA Receptor α1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and α1 Subunit Protein Expression

Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABAA receptor α1 subunit mutation (α1A322D). GABAA receptor αβγ subunits are arranged around the pore in a β-α-β-α-γ sequence (counterclockwise from the synaptic cleft). Therefore, each α1 subunit has different adjacent subunits, and heterozygous expression of α1(A322D), β, and γ subunits could produce receptors with four different subunit arrangements: β-α1-β-α1-γ (wild type); β-α1(A322D)-β-α1-γ (Hetβαβ); β-α1-β-α1(A322D)-γ (Hetβαγ);β-α1(A322D)-β-α1(A322D)-γ (homozygous). Expression of a 1:1 mixture of wild-type andα1(A322D) subunits with β2S and γ2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface α1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. α1(A322D) mutations were then made in covalently tethered triplet (γ2S-β2S-α1) and tandem (β2S-α1) concatamers to target selectively α1(A322D) to each of the asymmetric α1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Hetβαβ or Hetβαγ receptors. Hetβαβ currents were smaller than wild type and much larger than Hetβαγ and homozygous currents. Furthermore, Hetβαβ transfections contained less β-α concatamer than wild type but more than both Hetβαγ and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous α1(A322D)β2Sγ2S receptors depended on the position of the mutant α1 subunit, and GABAA receptor currents in heterozygous individuals likely result primarily from wild-type and Hetβαβ receptors with little contribution from Hetβαγ and homozygous receptors.

Decreased viability and absence-like epilepsy in mice lacking or deficient in the GABAA receptor α1 subunit.

Autosomal dominant mutations S326fs328X and A322D in the GABAA receptor α1 subunit are associated with human absence epilepsy and juvenile myoclonic epilepsy, respectively. Because these mutations substantially reduce α1 subunit protein expression in vitro, it was hypothesized that they produce epilepsy by causing α1 subunit haploinsufficiency. However, in a mixed background strain of mice, α1 subunit deletion does not reduce viability or cause visually apparent seizures; the effects of α1 subunit deletion on electroencephalography (EEG) waveforms were not investigated. Here, we determined the effects of α1 subunit loss on viability, EEG spike‐wave discharges and seizures in congenic C57BL/6J and DBA/2J mice. Deletion of α1 subunit caused strain‐ and sex‐dependent reductions in viability. Heterozygous mice experienced EEG discharges and absence‐like seizures within both background strains, and exhibited a sex‐dependent effect on the discharges and viability in the C57BL/6J strain. These findings suggest that α1 subunit haploinsufficiency can produce epilepsy and may be a major mechanism by which the S326fs328X and A322D mutations cause these epilepsy syndromes.

Structural Determinants of Benzodiazepine Allosteric Regulation of GABAA Receptor Currents

Benzodiazepine enhancement of GABAA receptor current requires a γ subunit, and replacement of the γ subunit by the δ subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABAA receptors at the junction between α and γ subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABAA receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using γ2L-δ GABAA receptor subunit chimeras coexpressed withα1 andβ3 subunits in human embryonic kidney 293T cells. Different domains of the γ2L subunit were replaced by δ subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2–M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2–M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2–M3 loop domains.

Correlation of Severity of FDG-PET Hypometabolism and Interictal Regional Delta Slowing in Temporal Lobe Epilepsy

Summary:  Purpose: We investigated the association of severity of hypometabolism detected by positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) and persistence of interictal EEG focal slowing in patients with refractory temporal lobe epilepsy.

GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors

A GABAA receptor (GABAAR) α1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused α1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual α1(AD) subunit expression on wild type GABAAR expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the α1(AD) subunit did not substitute for wild type α1 subunits on the cell surface, it reduced the surface expression of α1β2γ2 and α3β2γ2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The α1(AD) subunit reduced surface expression of α3β2γ2 receptors by a greater amount than α1β2γ2 receptors, thus altering cell surface GABAAR composition. When transfected into cultured cortical neurons, the α1(AD) subunit altered the time course of miniature inhibitory postsynaptic current kinetics and reduced miniature inhibitory postsynaptic current amplitudes. These findings demonstrated that, in addition to causing a heterozygous loss of function of α1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype.

Musicogenic seizures can arise from multiple temporal lobe foci : Intracranial EEG analyses of three patients

Summary:  Purpose: To determine the ictal‐onset zone of musicogenic seizures by using intracranial EEG monitoring.

GABAA Receptor Mutations Associated with Generalized Epilepsies1

Publisher Summary This chapter reviews the γ‐aminobutyric acid (GABA A ) receptor mutations associated with generalized epilepsies. GABA A receptors are the primary mediators of fast inhibitory synaptic transmission in the central nervous system (CNS) and are repeatedly documented to play a critical role in animal models of seizures. GABA A receptors are formed by pentameric assembly of multiple subunit subtypes. The most common GABA A receptors contain two α subunits, two β subunits, and a γ or δ subunit. Idiopathic generalized epilepsies (IGEs) are characterized by absence, myoclonic, and/or primary generalized tonic–clonic seizures in the absence of structural brain abnormalities and have a genetic basis. Mutations associated with IGEs are also identified in human GABA A receptor genes. The chapter focuses on the described human GABA A receptor channel epilepsy mutations. The mutations in GABA A receptor γ2, α1, and δ subunits that are associated with different IGE syndromes are reviewed in the chapter. These mutations alter GABA A receptor gating, expression, and/or trafficking of the receptor to the cell surface, all pathophysiological mechanisms that result in reduced GABA‐evoked currents that in neurons would cause neuronal disinhibition and thus predispose affected patients to manifest seizures.