Neuronal Inhibition in Epilepsy: Native Human GABA-A Receptors and Pharmacological Chaperones Restoring Expression of Disease Associated Variants

Abstract

Resolving Native GABA_A Receptor Structures From the Human Brain

Zhou J, Noviello CM, Teng J, Moore H, Lega B, Hibbs RE. Nature. 2025;638(8050):562–568. DOI: 10.1038/s41586-024-08454-1

Type A GABA (γ-aminobutyric acid) receptors (GABA_A receptors) mediate most fast inhibitory signalling in the brain and are targets for drugs that treat epilepsy, anxiety, depression and insomnia and for anaesthetics.^1,2 These receptors comprise a complex array of 19 related subunits, which form pentameric ligand-gated ion channels. The composition and structure of native GABA_A receptors in the human brain have been inferred from subunit localization in tissue,^1,3 functional measurements and structural analysis from recombinant expression^4–7 and in mice.⁸ However, the arrangements of subunits that co-assemble physiologically in native human GABA_A receptors remain unknown. Here we isolated α1 subunit-containing GABA_A receptors from human patients with epilepsy. Using cryo-electron microscopy, we defined a set of 12 native subunit assemblies and their 3D structures. We address inconsistencies between previous native and recombinant approaches, and reveal details of previously undefined subunit interfaces. Drug-like densities in a subset of these interfaces led us to uncover unexpected activity on the GABA_A receptor of antiepileptic drugs and resulted in localization of one of these drugs to the benzodiazepine-binding site. Proteomics and further structural analysis suggest interactions with the auxiliary subunits neuroligin 2 and GARLH4, which localize and modulate GABA_A receptors at inhibitory synapses. This work provides a structural foundation for understanding GABA_A receptor signalling and targeted pharmacology in the human brain.

Pharmacological Chaperones Restore Proteostasis of Epilepsy-Associated GABA_A Receptor Variants

Wang YJ, Seibert H, Ahn LY, Schaffer AE, Mu TW. Pharmacol Res. 2024 208, 107356 DOI: 10.1016/j.phrs.2024.107356

Recent advances in genetic diagnosis identified variants in genes encoding GABA_A receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABA_A receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the α1 variants. Mechanism of action study demonstrated that they enhance the folding, assembly, and trafficking and reduce the degradation of GABA_A variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABA_A receptor-specific manner.

Commentary

Management of epilepsy often relies on drugs that modulate GABA-A receptor (GABA_AR) activity, yet our understanding of the true molecular diversity, assembly, and pharmacology of these receptors in the human brain lags behind advances in animal models and recombinant systems, limiting translation into better therapies. Two recent articles represent landmark advances in understanding GABA_AR biology and clinical implications. Zhou et al provide the first high-resolution structural and compositional analysis of native human GABA_ARs, while Wang et al identified pharmacological chaperones for epilepsy-associated GABA_AR variants, offering a new strategy to rescue the function of disease-causing mutant receptors.^1,2

Paper 1

GABA_ARs show remarkable heterogeneity, with 19 distinct genes encoding various α, β, γ, δ, and other subunits that assemble to form a pentameric ligand-gated chloride channel. Until now, our knowledge of GABA_AR architecture has been largely based on recombinant expression systems and rodent models. ³ Zhou et al used affinity purification, mass spectrometry, and cryo-electron microscopy on resected brain tissue from epilepsy patients to isolate and characterize α1-containing GABA_ARs. Their analysis revealed 12 distinct pentameric assemblies containing various combinations of α1, α2, α3, β1, β2, β3, and γ2 subunits. The most common assembly was β2–α1–β2–α1–γ2, confirming previous predictions. They also discovered novel assemblies with mixed β2 and β3, and α2 and α3 subunits, revealing a unique diversity of human GABA_ARs. This is important, as GABA_AR subunit composition determines receptor function, pharmacology, and synaptic localization. ³ For the first time, β3 subunits were found to co-assemble with α1 in native human receptors; a composition not found in model organisms. Together, these findings highlight species- and tissue-specific diversity, providing a structural rationale for functional heterogeneity observed in pharmacological studies, for example, γ2 subunits are necessary for benzodiazepine binding. ⁴

A striking finding is the discovery of a potentially additional mechanism of action for lamotrigine. ¹ Cryo-electron microscopy revealed drug-like densities at α1–γ2 benzodiazepine binding site in several assemblies. Out of 6 antiseizure drugs taken by the tissue donors, lamotrigine was confirmed to bind to this site. In electrophysiological experiments, a clinically relevant concentration ⁵ of lamotrigine showed positive allosteric modulation of overexpressed GABA_ARs receptors containing γ2 in HEK293 cells. This is a paradigm-shifting discovery, as lamotrigine was previously thought to act almost exclusively via sodium channel blockade. ⁶

The discovery of novel subunit assemblies not seen in animal models highlights the limitations of extrapolating data from rodents to humans, especially for drug discovery. The finding that lamotrigine modulates GABA_AR function may facilitate the development of a new class of GABAergic modulators and infers that other clinically used drugs may have unrecognized actions at human receptors. These findings underscore the need for caution when interpreting preclinical data and reinforce the importance of human-based research. This work brilliantly demonstrates how cutting-edge structural and proteomic science can unravel longstanding mysteries of receptor biology.

While this study focused on α1-containing receptors from epilepsy patients, it opens the door for future work to explore other important subunit combinations, such as δ-containing receptors involved in tonic inhibition. ⁴ The GABA_AR subtypes identified in this study may not fully capture the diversity present in healthy brains or other disease contexts, as factors such as epilepsy and treatment history can influence receptor composition. In rodents, for example, both lamotrigine and benzodiazepines have been shown to alter subunit expression.^7–9 And clinical impacts of lamotrigine's binding remain open. Still, this study highlights the importance of studying human brain tissue to accelerate clinical translation.

Paper 2

So far, over 150 epilepsy-associated mutations have been identified in >10 out of 19 genes encoding GABA_AR subunits. ¹⁰ These mutations often lead to misfolded or improperly assembled receptors, resulting in trafficking deficits that cause diminished inhibitory signalling and increased seizure susceptibility. Increasing the number of GABA_ARs at the plasma membrane, even if their chloride conductance is reduced, would render antiepileptic drugs that enhance GABAergic transmission more effective. There are 2 ways that small molecules can increase surface expression. ² Proteostasis regulators affect synthesis, folding, localization, and degradation by reprogramming these cellular processes, but widely affect the trafficking of many proteins. However, pharmacological chaperones directly bind to their target proteins, stabilize them, and thereby specifically improve their trafficking to their functional location.

Wang et al systematically analyzed a panel of epilepsy-associated GABA_ARs with mutations in the α1 subunit, revealing that many are retained in the endoplasmic reticulum, resulting in reduced surface expression and function. ² To find pharmacological chaperones to rescue GABA_AR function, the authors then screened the effects of known GABA_AR modulators for rescue of cell surface expression and GABA-evoked currents of these receptors. Hispidulin and TP003 were identified to largely restore surface expression and the function of all or most of the mutant receptors tested in both cell lines and human iPSC-derived neurons. Importantly, these chaperones rescued both trafficking and function of several loss-of-function variants containing mutations in the α1 subunit. Since hispidulin and TP003 are both known positive allosteric modulators of GABA_ARs, their use may not only restore the trafficking of GABA_AR mutants, but also enhance their own and other GABA_AR-mediated currents once the receptors reach the plasma membrane, offering dual therapeutic benefits. Boosting receptor surface expression by stabilizing folding and assembly is critical, as many misfolded GABA_AR variants accumulate in the endoplasmic reticulum (ER), triggering ER stress, cell death, and a plethora of network-level dysfunctions underlying severe epilepsy phenotypes.

This work demonstrates a clear path toward precision medicine in epilepsy, where therapies are tailored to the specific molecular defect in each patient. The ability to rescue the function of several misfolded or improperly assembled GABA_ARs with pharmacological chaperones represents a major advance over traditional approaches that simply enhance GABAergic tone. This raises the possibility of genotype-guided therapy for patients with refractory epilepsy involving GABA_AR mutations. Bringing these chaperones into clinical use has its challenges, but it represents an exciting frontier. Though most evidence is still from in vitro and animal studies, this tailored treatment strategy to receptor subtype and mutation offers real promise for overcoming the limitations of current therapies.

These landmark studies are propelling epilepsy research into a new era, bringing the true molecular diversity of human GABA_ARs into focus and making therapies for genetic epilepsies a realistic goal. By uncovering previously unknown receptor assemblies and mechanisms, such as lamotrigine's direct modulation of GABA_ARs, and demonstrating that pharmacological chaperones can restore function to disease-causing variants, this work illustrates the impact of human-based basic science on therapeutic innovation. Notably, the success of pharmacological chaperones in rescuing GABA_AR variants also highlights their broader potential to correct misfolding and ER retention in a range of other protein misfolding diseases. By charting these new territories, both studies set the stage for further discoveries in GABA_AR biology and therapeutic innovation.