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Structural basis of GABARAP-mediated GABA_A receptor trafficking and functions on GABAergic synaptic transmission.

ABSTRACT: GABA_A receptors (GABA_ARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABA_ARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABA_ARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously unappreciated region in the ?2 subunit of GABA_AR. We demonstrate that GABARAP functions to stabilize GABA_ARs via promoting its trafficking pathway instead of blocking receptor endocytosis. The GABARAPL1-?2-GABA_AR crystal structure reveals the mechanisms underlying the complex formation. We provide evidence showing that phosphorylation of ?2-GABA_AR differentially modulate the receptor's binding to GABARAP and the clathrin adaptor protein AP2. Finally, we demonstrate that GABAergic synaptic currents are reduced upon specific blockage of the GABARAP-GABA_AR complex formation. Collectively, our results reveal that GABARAP/GABARAPL1, but not other members of the Atg8 family proteins, specifically regulates synaptic localization of GABA_ARs via modulating the trafficking of the receptor.

SUBMITTER: Ye J

PROVIDER: S-EPMC7803741 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Structural basis of GABARAP-mediated GABAA receptor trafficking and functions on GABAergic synaptic transmission.

Ye Jin J Zou Guichang G Zhu Ruichi R Zhu Ruichi R Kong Chao C Miao Chenjian C Zhang Mingjie M Li Jianchao J Xiong Wei W Wang Chao C

Nature communications 20210112 1

GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABAARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABAARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously ...[more]

PMID: 33436612

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Project description:BackgroundDisruption of normal brain development is implicated in numerous psychiatric disorders with neurodevelopmental origins, including autism spectrum disorder (ASD). Widespread abnormalities in brain structure and functions caused by dysregulations of neurodevelopmental processes has been recently shown to exert adverse effects across generations. An imbalance between excitatory/inhibitory (E/I) transmission is the putative hypothesis of ASD pathogenesis, supporting by the specific implications of inhibitory γ-aminobutyric acid (GABA)ergic system in autistic individuals and animal models of ASD. However, the contribution of GABAergic system in the neuropathophysiology across generations of ASD is still unknown. Here, we uncover profound alterations in the expression and function of GABAA receptors (GABAARs) in the amygdala across generations of the VPA-induced animal model of ASD.MethodsThe F2 generation was produced by mating an F1 VPA-induced male offspring with naïve females after a single injection of VPA on embryonic day (E12.5) in F0. Autism-like behaviors were assessed by animal behavior tests. Expression and functional properties of GABAARs and related proteins were examined by using western blotting and electrophysiological techniques.ResultsSocial deficit, repetitive behavior, and emotional comorbidities were demonstrated across two generations of the VPA-induced offspring. Decreased synaptic GABAAR and gephyrin levels, and inhibitory transmission were found in the amygdala from two generations of the VPA-induced offspring with greater reductions in the F2 generation. Weaker association of gephyrin with GABAAR was shown in the F2 generation than the F1 generation. Moreover, dysregulated NMDA-induced enhancements of gephyrin and GABAAR at the synapse in the VPA-induced offspring was worsened in the F2 generation than the F1 generation. Elevated glutamatergic modifications were additionally shown across generations of the VPA-induced offspring without generation difference.ConclusionsTaken together, these findings revealed the E/I synaptic abnormalities in the amygdala from two generations of the VPA-induced offspring with GABAergic deteriorations in the F2 generation, suggesting a potential therapeutic role of the GABAergic system to generational pathophysiology of ASD.

Project description:BackgroundPrevious reports of inhibition in the neocortex suggest that inhibition is mediated predominantly through GABA(A) receptors exhibiting fast kinetics. Within the hippocampus, it has been shown that GABA(A) responses can take the form of either fast or slow response kinetics. Our findings indicate, for the first time, that the neocortex displays synaptic responses with slow GABA(A) receptor mediated inhibitory postsynaptic currents (IPSCs). These IPSCs are kinetically and pharmacologically similar to responses found in the hippocampus, although the anatomical specificity of evoked responses is unique from hippocampus. Spontaneous slow GABA(A) IPSCs were recorded from both pyramidal and inhibitory neurons in rat visual cortex.ResultsGABA(A) slow IPSCs were significantly different from fast responses with respect to rise times and decay time constants, but not amplitudes. Spontaneously occurring GABA(A) slow IPSCs were nearly 100 times less frequent than fast sIPSCs and both were completely abolished by the chloride channel blocker, picrotoxin. The GABA(A) subunit-specific antagonist, furosemide, depressed spontaneous and evoked GABA(A) fast IPSCs, but not slow GABA(A)-mediated IPSCs. Anatomical specificity was evident using minimal stimulation: IPSCs with slow kinetics were evoked predominantly through stimulation of layer 1/2 apical dendritic zones of layer 4 pyramidal neurons and across their basal dendrites, while GABA(A) fast IPSCs were evoked through stimulation throughout the dendritic arborization. Many evoked IPSCs were also composed of a combination of fast and slow IPSC components.ConclusionGABA(A) slow IPSCs displayed durations that were approximately 4 fold longer than typical GABA(A)fast IPSCs, but shorter than GABA(B)-mediated inhibition. The anatomical and pharmacological specificity of evoked slow IPSCs suggests a unique origin of synaptic input. Incorporating GABA(A) slow IPSCs into computational models of cortical function will help improve our understanding of cortical information processing.

Project description:Synapsins are epilepsy susceptibility genes that encode phosphoproteins reversibly associated with synaptic vesicles. Synapsin II (SynII) gene deletion produces a deficit in inhibitory synaptic transmission, and this defect is thought to cause epileptic activity. We systematically investigated how SynII affects synchronous and asynchronous release components of inhibitory transmission in the CA1 region of the mouse hippocampus. We found that the asynchronous GABAergic release component is diminished in SynII-deleted (SynII(-)) slices. To investigate this defect at different interneuron subtypes, we selectively blocked either N-type or P/Q-type Ca2+ channels. SynII deletion suppressed the asynchronous release component at synapses dependent on N-type Ca2+ channels but not at synapses dependent on P/Q-type Ca2+ channels. We then performed paired double-patch recordings from inhibitory basket interneurons connected to pyramidal neurons and used cluster analysis to classify interneurons according to their spiking and synaptic parameters. We identified two cell subtypes, presumably parvalbumin (PV) and cholecystokinin (CCK) expressing basket interneurons. To validate our interneuron classification, we took advantage of transgenic animals with fluorescently labeled PV interneurons and confirmed that their spiking and synaptic parameters matched the parameters of presumed PV cells identified by the cluster analysis. The analysis of the release time course at the two interneuron subtypes demonstrated that the asynchronous release component was selectively reduced at SynII(-) CCK interneurons. In contrast, the transmission was desynchronized at SynII(-) PV interneurons. Together, our results demonstrate that SynII regulates the time course of GABAergic release, and that this SynII function is dependent on the interneuron subtype.SIGNIFICANCE STATEMENT Deletion of the neuronal protein synapsin II (SynII) leads to the development of epilepsy, probably due to impairments in inhibitory synaptic transmission. We systematically investigated SynII function at different subtypes of inhibitory neurons in the hippocampus. We discovered that SynII affects the time course of GABA release, and that this effect is interneuron subtype specific. Within one of the subtypes, SynII deficiency synchronizes the release and suppresses the asynchronous release component, while at the other subtype SynII deficiency suppresses the synchronous release component. These results reveal a new SynII function in the regulation of the time course of GABA release and demonstrate that this function is dependent on the interneuron subtype.

Project description:Key pointsCurrent views suggest ?2 subunit-containing GABAA receptors mediate phasic IPSCs while extrasynaptic ? subunits mediate diffusional IPSCs and tonic current. We have re-examined the roles of the two receptor populations using mice with picrotoxin resistance engineered into receptors containing the ? subunit. Using pharmacological separation, we find that in general ? and ? IPSCs are modulated in parallel by manipulations of transmitter output and diffusion, with evidence favouring modestly more diffusional contribution to ? IPSCs. Our findings also reveal that spontaneous ? IPSCs are mainly driven by channel deactivation, rather than by diffusion of GABA. Understanding the functional contributions of the two receptor classes may help us understand the actions of drug therapies with selective effects on one population over the other.AbstractGABAA receptors mediate transmission throughout the central nervous system and typically contain a ? subunit (? receptors) or a ?2 subunit (?2 receptors). ? IPSCs decay slower than ?2 IPSCs, but the reasons are unclear. Transmitter diffusion, rebinding, or slow deactivation kinetics of channels are candidates. We used gene editing to confer picrotoxin resistance on ? receptors in mice, then pharmacologically isolated ? receptors in mouse dentate granule cells to explore IPSCs. ?2 and ? components of IPSCs were modulated similarly by presynaptic manipulations and manipulations of transmitter lifetime, suggesting that GABA release recruits ? receptors proportionally to ?2 receptors. ? IPSCs showed more sensitivity to altered transmitter release and to a rapidly dissociating antagonist, suggesting an additional spillover contribution. Reducing GABA diffusion with 5% dextran increased the peak amplitude and decreased the decay of evoked ? IPSCs but had no effect on ? or dual-component (mainly ?2-driven) spontaneous IPSCs, suggesting that GABA actions can be local for both receptor types. Rapid application of varied [GABA] onto nucleated patches from dentate granule cells demonstrated a deactivation rate of ? receptors similar to that of ? spontaneous IPSCs, consistent with the idea that deactivation and local GABA actions drive ? spontaneous IPSCs. Overall, our results indicate that ? IPSCs are activated by both synaptic and diffusional GABA. Our results are consistent with a functional relationship between ? and ?2 GABAA receptors akin to that of slow NMDA and fast AMPA EPSCs at glutamate synapses.

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Structural basis of GABARAP-mediated GABA_A receptor trafficking and functions on GABAergic synaptic transmission.

Publications

Structural basis of GABARAP-mediated GABA<sub>A</sub> receptor trafficking and functions on GABAergic synaptic transmission.

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