Highlights d Acute high-fat-diet feeding activates PNOC neurons in the arcuate nucleus (ARC) d GABAergic PNOC ARC neurons inhibit anorexigenic POMC neurons d Optogenetic activation of PNOC ARC neurons promotes feeding d Ablation of PNOC ARC neurons protects from obesity
GABA A receptors (GABA A Rs) are the principal inhibitory neurotransmitter receptors in the central nervous system. They control neuronal excitability by synaptic and tonic forms of inhibition mostly mediated by different receptor subtypes located in specific cell membrane subdomains. A consensus suggests that α1-3βγ comprise synaptic GABA A Rs, whilst extrasynaptic α4βδ, α5βγ and αβ isoforms largely underlie tonic inhibition. Although some structural features that enable the spatial segregation of receptors are known, the mobility of key synaptic and extrasynaptic GABA A Rs are less understood, and yet this is a key determinant of the efficacy of GABA inhibition. To address this aspect, we have incorporated functionally silent α-bungarotoxin binding sites (BBS) into prominent hippocampal GABA A R subunits which meidate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABA A Rs that are traditionally considered to mediate synaptic or tonic inhibition are all able to access inhibitory synapses. These isoforms have variable diffusion rates and are differentially retained upon entering the synaptic membrane subdomain. Interestingly, α2 and α4 subunits reside longer at synapses compared to α5 and δ subunits. Furthermore, a high proportion of extrasynaptic δ-containing receptors exhibited slower diffusion compared to δ subunits at synapses. A chimera formed from δ-subunits, with the intracellular domain of γ2L, reversed this behaviour. In addition, we observed that receptor activation affected the diffusion of extrasynaptic, but not of synaptic GABA A Rs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABA A Rs are determined by receptor subunit composition and intracellular structural motifs. Words -248Highlights 1. GABA A Rs mediating synaptic or tonic inhibition all access inhibitory synapses 2. Diffusion and retention of GABA A Rs at synapses depends on the subunit composition 3. Dwell times for α2 and α4 are longer than for α5 and δ at inhibitory synapses 4. A large proportion of extrasynaptic δ-GABA A Rs exhibit restricted diffusion 5. The large intracellular loops of δ and γ2L regulate mobility and synaptic trapping
GABA A receptors (GABA A Rs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren Syndrome identified a frameshifting de novo variant in a major GABA A R gene, GABRA1. This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1 Lys374Serfs*25. Cell surface expression of mutant murine GABA A Rs is severely impaired compared to wild-type, due to retention in the endoplasmic reticulum. Mutant receptors were differentially co-expressed with β3, but not with β2 subunits in mammalian cells. Reduced surface expression was reflected by smaller inhibitory postsynaptic currents, which may underlie the induction of seizures. The mutant does not have a dominant negative effect on native neuronal GABA A R expression since GABA current density was unaffected in hippocampal neurons, even though mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential β subunit expression of GABA A R populations, which profoundly affected receptor function and synaptic inhibition. Significance Statement GABA A Rs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain and their dysfunction underlies many neurological disorders, especially epilepsy. Here we report the characterisation of an α1-GABA A R variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1-subunit cell surface expression with β subunits resulting in severely reduced synaptic inhibition. Our study reveals that diseaseinducing variants can affect GABA A R structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.
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