Presynaptic hyperexcitability reversed by positive allosteric modulation of a GABABR epilepsy variant.

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, I705N) have been identified in the gene (GABBR2) encoding for GABABR2. Individuals that harbour 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 modelling, biochemistry, live cell Ca2+ imaging of presynaptic terminals, whole-cell electrophysiology of HEK-293T cells and neurons, and two-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 three point mutations impair neuronal cell surface expression of GABABRs, reducing signalling efficacy. However, a negative effect evident for one variant perturbed neurotransmission by elevating presynaptic Ca2+ signalling. This is reversed by enhancing GABABR signalling 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 whilst highlighting the translational suitability and therapeutic potential of allosteric modulation for correcting these deficits.

Forty Years Searching for Neurosteroid Binding Sites on GABAA Receptors.

Brain inhibition is a vital process for controlling and sculpting the excitability of the central nervous system in healthy individuals. This level of control is provided over several timescales and involves the neurotransmitter GABA acting at inhibitory synapses to: rapidly inhibit neurons by activating the GABAA receptor; over a slower timescale, to tonically activate extrasynaptic GABAA receptors to provide a low level of background inhibition; and finally, to activate G-protein coupled GABAB receptors to control transmitter release by inhibiting presynaptic Ca2+ channels whilst providing postsynaptic inhibition via K+ channel activation. From this plethora of roles for GABA and its receptors, the GABAA receptor isoform is of major interest due to its dynamic functional plasticity, which in part, is due to being targeted by modulatory brain neurosteroids derived from sex and stress hormones. This family of neurosteroids can, depending on their structure, potentiate, activate and also inhibit the activity of GABAA receptors to affect brain inhibition. This review tracks the methods that have been deployed in probing GABAA receptors, and charts the sterling efforts made by several groups to locate the key neurosteroid binding sites that affect these important receptors. Increasing our knowledge of these binding sites will greatly facilitate our understanding of the physiological roles of neurosteroids and will help to advance their use as novel therapeutics to combat debilitating brain diseases.

Mechanisms of inhibition and activation of extrasynaptic αß GABAA receptors.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, ß-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αß, α4ßδ, α6ßδ and α5ßγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1ßγ, α2ßγ and α3ßγ receptor responses5,7-12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αß GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA-Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αß receptors that adapt them to a role in tonic signalling.

Physiological role for GABAA receptor desensitization in the induction of long-term potentiation at inhibitory synapses.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.