Shifting Focus: The “Other” GABAR

Abstract

Presynaptic Hyperexcitability Reversed by Positive Allosteric Modulation of a GABABR Epilepsy Variant

Marielle Minere, Martin Mortensen, Valentina Dorovykh, Gary Warnes, Dean Nizetic, Trevor G Smart, Saad B Hannan. Brain. 2024: awae232. doi/10.1093/brain/awae232/7717204

Gamma-aminobutyric acid B receptors (GABABRs) are key membrane proteins that continually adapt the excitability of the nervous system. These G-protein-coupled receptors are activated by the brain's premier inhibitory neurotransmitter GABA. They are obligate heterodimers composed of GABA-binding GABABR1 and G-protein-coupling GABABR2 subunits. Recently, three variants (G693W, S695I, and I705N) have been identified in the gene (GABBR2) encoding for GABABR2. Individuals that harbor any of these variants exhibit severe developmental epileptic encephalopathy and intellectual disability, but the underlying pathogenesis that is triggered in neurons remains unresolved. Using a range of confocal imaging, flow cytometry, structural modeling, biochemistry, live cell Ca²⁺ imaging of presynaptic terminals, whole-cell electrophysiology of HEK-293T cells and neurons, and 2-electrode voltage clamping of Xenopus oocytes we have probed the biophysical and molecular trafficking and functional profiles of G693W, S695I, and I705N variants.We report that all 3-point mutations impair neuronal cell surface expression of GABABRs, reducing signaling efficacy. However, a negative effect is evident for 1 variant perturbed neurotransmission by elevating presynaptic Ca²⁺ signaling. This is reversed by enhancing GABABR signaling via positive allosteric modulation.Our results highlight the importance of studying neuronal receptors expressed in nervous system tissue and provide new mechanistic insights into how GABABR variants can initiate neurodevelopmental disease while highlighting the translational suitability and therapeutic potential of allosteric modulation for correcting these deficits.

Commentary

The role of gamma-aminobutyric acid (GABA) receptors (GABAR) in development and disease is commonly studied and reported for GABA A receptors (GABAARs). However, GABA B receptors (GABABRs) have remained, in comparison, less investigated in this context despite the clear involvement they have in several processes including seizure susceptibility. The GABABR is the metabotropic receptor of GABA, the main inhibitory neurotransmitter in the mammalian central nervous system. GABABR is a 7 transmembrane domain-containing protein belonging to class C, G-protein-coupled receptors (GPCRs) and is functional as a heterodimer containing GABAB1 and GABAB2 subunits. Only the GABAB1 subunit can bind agonists and contains the GABA binding site, whereas the GABAB2 subunit is responsible for G-protein coupling. Since GABABRs are obligate heterodimers, oligomerization is indispensable for cell surface insertion, stability, and function. GABABR has multipronged pre- and postsynaptic modulations known to have important effects on depression and memory. ¹ As a GPCR, GABABR is the pivot of neurobiological mechanisms underlying emotion, memory, nociception, movement, and psychiatric phenotypes. The best-known neuronal GABABR functions are the gating of voltage-sensitive Ca²⁺ (Cav) channels and inwardly rectifying Kir3-type K+ channels by the Gβγ subunits of the G protein that mediate slow synaptic inhibition in the central nervous system. Neuronal GIRK (G-protein-gated inwardly rectifying potassium) channels are formed by homo- and hetero-assembly among GIRK1, GIRK2, and GIRK3 subunits and play a critical role in modulating neuronal excitability throughout the brain. GABABR-GIRK signaling mediates postsynaptic inhibition of neuronal firing. This indicates the potential usefulness of GABAB receptor stimulation as a therapeutic target in several medical conditions including epilepsy.

This study investigates three variants (G693W, S695I, and I705N) identified in the gene (GABBR2) encoding for GABABR2. ² Twelve individuals with any of these variants presented with severe developmental epileptic encephalopathy and intellectual disability (ID). The evolution of the mechanisms and pathogenesis in neurons underlying these mutations remains poorly understood. Clinical outcomes are commonly diagnosed as epileptic encephalopathy, infantile or early childhood, 2(IECEE2; DEE92)^3,4; sometimes associated with ID. Developmental and epileptic encephalopathy-92 (DEE92) is characterized by the onset of seizures in infancy or childhood and associated with global developmental delay and variable impairment of intellectual development. Severely affected patients are unable to walk or speak, and neurological features can include cortical blindness, dystonia, and spasticity. ⁵ The GABABR2 subunit is critical for the cell surface trafficking of GABABRs, which determines the functional efficacy of GABAergic inhibition. Since GABABR2 variants could orchestrate seizures and neurodevelopmental deficits due to reduced neuronal GABABR signaling, previous studies were undertaken to investigate knocking out GABABR2, testing GABABR antagonist treatment, or impairing GABABR cell surface delivery. All such manipulation resulted in the emergence of seizures. Using several approaches, the authors of this study ² report severe impairment of neuronal plasma membrane expression of the variants associated with deficits in presynaptic Ca²⁺ signaling. A GABABR-positive allosteric modulator (PAM) rescued the synaptic deficits detected in their phenotypic characterization.

The GABABR2 variants of highly conserved sites G693, S695, and I705 were shown to align at the heterodimer interface of the activated receptor. Whole-cell electrophysiology was used to measure GABA-activated GIRK currents by sequentially expressing GABABR2 variants with GABABR1 to promote heteromeric cell surface expression in GIRK cells (HEK-293T cells expressing Kir3.1/3.2 channels). Analysis of the maximal GABA-activated K+ currents revealed reduced maxima for both G693W and I705N. S695I yielded negligible current associated with severe functional impairment. Previous enzyme-liked immunosorbent assay (ELISA) or immunofluorescence studies had concluded that I705N expression remained unaffected; however, the flow cytometry method used in this study detected a reduction in expression. G693W and S695I expression were well-maintained at similar levels compared to wild-type receptors indicating that the absence of K+ currents could be due to additional signaling-dependent deficits. The functional consequences of reduced neuronal cell membrane GABABR expression were assessed in hippocampal neurons expressing wild-type or enhanced green fluorescent protein (eGFP)-tagged variant GABABR2. GABABR agonist baclofen was used to activate GIRK currents in these neurons. S695I and I705N showed a significant reduction in GABABR function. S695I variant receptors, additionally, could suppress wild-type GABABR function as well, adding mechanistic insights to the absence of K+ currents in previous assays. Biochemical evidence from immunoprecipitation did not reveal changes to heterodimerization for S695I and therefore the mutation likely suffers from defective transduction of ligand-binding signals. Surprisingly, GABA potency at G693W and I705N receptors was increased compared to wild-type receptors (lower EC50s). Structural modeling tentatively indicated that this may be due to re-positioned R2-variant transmembrane domains (TMD). By studying GABABR2 variants in neurons, trafficking defects in GABABR2 were detected as a principal mechanism by which these receptors could cause dysfunction. A difference in cell surface expression for the R2 variant receptors when expressed in neurons compared to heterologous cells highlights differential quality control checks in neurons required for cell surface expression. Cryo-electron microscopy studies of the structure of GABABRs show different conformations while unbound and bound to agonists, modulators, and G protein. ⁶ TM6 is deemed crucial for receptor activation and forms an integral part of GS39783 and rac-BHFF PAM binding pockets. When expressed on the cell surface, N-terminal variants (R212Q, T394M, and G440R) are likely to affect GABA binding and the activation of signal transduction. Variants located within TM4 (A567T), TM5 (M668L), and TM6 (G693W, S695I, I705N, and A707T) affect signal transduction and G-protein coupling. Therefore, a diverse set of mechanisms can result in variant phenotypes.

GABABR candidate PAMs are conspicuously missing from the current list of small molecules in development for neurological disorders. ⁷ Additionally, no allosteric modulators for G-protein-coupled receptors have been approved for the treatment of psychiatric or neurological disorders, even though several allosteric modulators entered Phase II trials. ⁸ Despite the well-documented involvement of GABABR in disease, only 2 agonist drugs are currently on the market. Agonist baclofen (Lioresal^®) is prescribed for muscle rigidity and spasms associated with multiple sclerosis and spasticity and sodium oxybate, (Xyrem^®), to decrease daytime sleepiness and reduce sudden attacks of weak/paralyzed muscles (cataplexy) in narcoleptic patients. Proteomic approaches have revealed that native GABABR complexes comprise ∼30 proteins that provide a molecular basis for the functional diversity observed with these receptors. ⁹ Such increased understanding supports major advantages and the broad range of activities that can be therapeutically targeted through selective modulation of specific signaling pathways. On the flip side, targeting GABABRs for epilepsy is deemed complicated since GABABR modulation has been shown to be both pro- and anticonvulsant depending on the nature of the pathological neuronal networks involved. ¹⁰ Preclinical data reported that variant-induced presynaptic GABABR signaling defects were reversed by PAMs. However, these data so far have not been translated into clinical trials because of the potential for downstream adverse effects due to unknowns related to the GABABR native complex and associated proteins that define its functional diversity. The therapeutic goal for the use of new compounds in epilepsy awaits a strategy that targets GABABRs in specific pathological states.

Although the developmental switch between excitatory and inhibitory GABA-mediated responses is widely appreciated, the fact that the postnatal maturation of the GABAergic system lasts until late adolescence in humans is not persuasively promoted. ¹¹ The developmental profile and natural history of many neuropsychiatric disorders in the teenage years and early twenties provides a strong rationale to focus on the continued role of GABA in brain maturation. The role of GABABRs in FragileX ¹² and Rett syndrome ² highlights their role in early-life brain maturation. Given the role of GABABRs in behaviors underlying psychiatric disorders, GABABRs have not received the same attention as other targets in this context. ⁶ GABABR-focused research should help garner additional attention from experts in the field of epilepsy and psychiatry to develop GABABR-targeted therapies with the added focus on understanding the complexity of pathological states of GABARs by network and pathway specificity.