Ionotropic neurotransmitter receptors mediate fast synaptic transmission by functioning as ligand-gated ion channels. Fast inhibitory transmission in the brain is mediated mostly by ionotropic GABAA ...receptors (GABAARs), but their essential components for synaptic localization remain unknown. Here, we identify putative auxiliary subunits of GABAARs, which we term GARLHs, consisting of LH4 and LH3 proteins. LH4 forms a stable tripartite complex with GABAARs and neuroligin-2 in the brain. Moreover, LH4 is required for the synaptic localization of GABAARs and inhibitory synaptic transmission in the hippocampus. Our findings propose GARLHs as the first identified auxiliary subunits for anion channels. These findings provide new insights into the regulation of inhibitory transmission and the molecular constituents of native anion channels in vivo.
•The γ2-containing GABAARs form a tripartite complex with LH4 and neuroligin-2•GARLHs are GABAAR putative auxiliary subunits consisting of LH3 and LH4•The GABAAR γ2 subunit regulates protein expressions of LH4 and neuroligin-2•GARLH is required for the synaptic GABAAR localization and inhibitory transmission
Yamasaki et al. report GARLH family proteins as putative auxiliary subunits of GABAARs in vivo. GARLH forms a tripartite complex with GABAARs and neuroligin-2 and regulates the synaptic localization of GABAARs and inhibitory synaptic transmission.
Pore‐forming subunits of ion channels show channel activity in heterologous cells. However, recombinant and native channels often differ in their channel properties. These discrepancies are resolved ...by the identification of channel auxiliary subunits. In this review article, an auxiliary subunit of ligand‐gated ion channels is defined using four criteria: (1) as a Non‐pore‐forming subunit, (2) direct and stable interaction with a pore‐forming subunit, (3) modulation of channel properties and/or trafficking in heterologous cells, (4) necessity in vivo. We focus particularly on three classes of ionotropic glutamate receptors and their transmembrane interactors. Precise identification of auxiliary subunits and reconstruction of native glutamate receptors will open new directions to understanding the brain and its functions.
Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors ...mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and kainate type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively charged lipid bilayers in a phosphorylation-dependent manner and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.
► TARP phosphorylation regulates synaptic AMPA receptors in vivo ► TARP interacts with negatively charged lipids via electrostatic interactions ► Interaction of TARPs with lipids inhibits TARP binding to PSD-95 ► Dissociation of TARPs from lipid bilayers enhances synaptic AMPA receptor activity
Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the ...release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.
GABAA receptor (GABAAR) pentamers are assembled from a pool of 19 subunits, and variety in subunit combinations diversifies GABAAR functions to tune brain activity. Pentamers with distinct subunit ...compositions localize differentially at synaptic and non-synaptic sites to mediate phasic and tonic inhibition, respectively. Despite multitudes of theoretical permutations, limited subunit combinations have been identified in the brain. Currently, no molecular model exists for combinatorial GABAAR assembly in vivo. Here, we reveal assembly rules of native GABAAR complexes that explain GABAAR subunit subcellular distributions using mice and Xenopus laevis oocytes. First, α subunits possess intrinsic signals to segregate into distinct pentamers. Second, γ2 is essential for GABAAR assembly with Neuroligin-2 (NL2) and GARLHs, which localize GABAARs at synapses. Third, δ suppresses α6 synaptic localization by preventing assembly with GARLHs/NL2. These findings establish the first molecular model for combinatorial GABAAR assembly in vivo and reveal an assembly pathway regulating GABAAR synaptic localization.
Ionotropic neurotransmitter receptors at postsynapses mediate fast synaptic transmission upon binding of the neurotransmitter. Post- and trans-synaptic mechanisms through cytosolic, membrane, and ...secreted proteins have been proposed to localize neurotransmitter receptors at postsynapses. However, it remains unknown which mechanism is crucial to maintain neurotransmitter receptors at postsynapses. In this study, we ablated excitatory or inhibitory neurons in adult mouse brains in a cell-autonomous manner. Unexpectedly, we found that excitatory AMPA receptors remain at the postsynaptic density upon ablation of excitatory presynaptic terminals. In contrast, inhibitory GABA
receptors required inhibitory presynaptic terminals for their postsynaptic localization. Consistent with this finding, ectopic expression at excitatory presynapses of neurexin-3 alpha, a putative trans-synaptic interactor with the native GABA
receptor complex, could recruit GABA
receptors to contacted postsynaptic sites. These results establish distinct mechanisms for the maintenance of excitatory and inhibitory postsynaptic receptors in the mature mammalian brain.
Mossy cells (MCs) of the dentate gyrus are key components of an excitatory associative circuit established by reciprocal connections with dentate granule cells (GCs). MCs are implicated in place ...field encoding, pattern separation, and novelty detection, as well as in brain disorders such as temporal lobe epilepsy and depression. Despite their functional relevance, little is known about the determinants that control MC activity. Here, we examined whether MCs express functional kainate receptors (KARs), a subtype of glutamate receptors involved in neuronal development, synaptic transmission, and epilepsy. Using mouse hippocampal slices, we found that bath application of submicromolar and micromolar concentrations of the KAR agonist kainic acid induced inward currents and robust MC firing. These effects were abolished in GluK2 KO mice, indicating the presence of functional GluK2-containing KARs in MCs. In contrast to CA3 pyramidal cells, which are structurally and functionally similar to MCs and express synaptic KARs at mossy fiber (MF) inputs (i.e., GC axons), we found no evidence for KAR-mediated transmission at MF-MC synapses, indicating that most KARs at MCs are extrasynaptic. Immunofluorescence and immunoelectron microscopy analyses confirmed the extrasynaptic localization of GluK2-containing KARs in MCs. Finally, blocking glutamate transporters, a manipulation that increases extracellular levels of endogenous glutamate, was sufficient to induce KAR-mediated inward currents in MCs, suggesting that MC-KARs can be activated by increases in ambient glutamate. Our findings provide the first direct evidence of functional extrasynaptic KARs at a critical excitatory neuron of the hippocampus.
Hilar mossy cells (MCs) are an understudied population of hippocampal neurons that form an excitatory loop with dentate granule cells. MCs have been implicated in pattern separation, spatial navigation, and epilepsy. Despite their importance in hippocampal function and disease, little is known about how MC activity is recruited. Here, we show for the first time that MCs express extrasynaptic kainate receptors (KARs), a subtype of glutamate receptors critically involved in neuronal function and epilepsy. While we found no evidence for synaptic KARs in MCs, KAR activation induced strong action potential firing of MCs, raising the possibility that extracellular KARs regulate MC excitability
and may also promote dentate gyrus hyperexcitability and epileptogenesis.
Long-term potentiation (LTP), a well-characterized form of synaptic plasticity, is believed to underlie memory formation. Hebbian, postsynaptically expressed LTP requires TARPγ-8 phosphorylation for ...synaptic insertion of AMPA receptors (AMPARs). However, it is unknown whether TARP-mediated AMPAR insertion alone is sufficient to modify behavior. Here, we report the development of a chemogenetic tool, ExSYTE (Excitatory SYnaptic Transmission modulator by Engineered TARPγ-8), to mimic the cytoplasmic interaction of TARP with the plasma membrane in a doxycycline-dependent manner. We use this tool to examine the specific role of synaptic AMPAR potentiation in amygdala neurons that are activated by fear conditioning. Selective expression of active ExSYTE in these neurons potentiates AMPAR-mediated synaptic transmission in a doxycycline-dependent manner, occludes synaptically induced LTP, and mimics freezing triggered by cued fear conditioning. Thus, chemogenetic controlling of the TARP-membrane interaction is sufficient for LTP-like synaptic AMPAR insertion, which mimics fear conditioning.
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•ExSYTE, a chemogenetic tool, can control protein-lipid electrostatic interactions•Dissociation of the TARP cytoplasmic domain from plasma membranes mimics AMPAR-LTP•A subthreshold conditioning induces c-Fos expression in the amygdala without freezing•ExSYTE expression in activated amygdala neurons reversibly controls freezing
A chemogenetic tool, ExSYTE, controls the interaction of TARP with plasma membranes and potentiates AMPAR-mediated transmission, thereby mimicking synaptically induced LTP. Selective expression of ExSYTE in activated neurons modifies behavior in a chemogenetic manner.
Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates ...of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.
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