Clptm1 Limits Forward Trafficking of GABAA Receptors to Scale Inhibitory Synaptic Strength

Ge, Yuan; Kang, Yunhee; Cassidy, Robert M.; Moon, Kyung‐Mee; Lewis, Renate; Wong, Rachel; Foster, Leonard J.; Craig, Ann Marie

doi:10.1016/j.neuron.2017.12.038

Cited by 63 publications

(82 citation statements)

References 66 publications

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“…This work revealed shifts toward γ 2-GABA A R association with new protein pathway networks associated with cell surface adhesion, intracellular junctions, synaptic plasticity, endocytosis & recycling and ubiquitination ( Figure 6, Table 3), confirming similar fluctuations in membrane and intracellular trafficking occur in vivo and in vitro after DZP. Recent inhibitory synapse proteomics studies have identified a number of new protein synaptic constituents or modulators of GABA A R function (Butko et al, 2013;Kang et al, 2014;Nakamura et al, 2016b;Uezu et al, 2016;Ge et al, 2018). We show here that proteins known to have roles in synaptic function and trafficking of membrane receptors show changes in their association with γ 2-receptors.…”

Section: Discussionmentioning

confidence: 60%

“…6, Table 3), confirming similar fluctuations in membrane and intracellular trafficking occur in vivo and in vitro after DZP. Recent inhibitory synapse proteomics studies have identified a number of new protein synaptic constituents or modulators of GABA A R function (66)(67)(68)(69)(70). We show here that proteins known to have roles in synaptic function and trafficking of membrane receptors show changes in their association with γ2-receptors.…”

Section: Discussionmentioning

confidence: 60%

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Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

Lorenz-Guertin

Bambino

Das

et al. 2019

Preprint

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Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABA A Rs). Here we define key molecular events impacting γ2 GABA A R and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABA A Rs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2 pH FAP). Live-imaging experiments using γ2 pH FAP GABA A R expressing neurons identified enhanced lysosomal targeting of surface GABA A Rs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between α2 and γ2 subunits within a GABA A R in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABA A Rs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABA A R interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABA A R synaptic availability via multiple dynamic trafficking processes.

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 60%

Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

Lorenz-Guertin

Bambino

Das

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…They play an essential role in the nervous system and include γaminobutyric acid type A receptors (GABA A Rs), nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine type-3 receptors (5HT 3 Rs), and glycine receptors (GlyRs) [14]. To reach their final destination, individual subunits undergo synthesis, folding and assembly into pentameric receptors in the ER and then go through the ER-to-Golgi transport, post Golgi transport and surface membrane insertion [15][16][17][18][19][20]. Disturbing any biogenesis step of these neuroreceptors could influence their surface expression level and the functional synaptic transmission, and impairment of the neurotransmission process can lead to related neurological diseases [13].…”

Section: Introductionmentioning

confidence: 99%

LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors

Zhang

2019

Biochemical and Biophysical Research Communications

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The endoplasmic reticulum-Golgi intermediate compartment aka LMAN1), which cycles between the endoplasmic reticulum (ER) and Golgi, is a known cargo receptor for a number of soluble proteins. However, whether LMAN1 plays a role as a trafficking factor in the central nervous system is largely unknown. Here, we determined the role of LMAN1 on endogenous protein levels of the Cys-loop superfamily of neuroreceptors, including gammaaminobutyric acid type A receptors (GABA A Rs), 5-hydroxytryptamine (serotonin) type 3 (5-HT 3 ) receptors, and nicotinic acetylcholine receptors (nAChRs). Knockdown of LMAN1 reduces the surface trafficking of endogenous β3 subunits of GABA A Rs in mouse hypothalamic GT1-7 neurons. Furthermore, Western blot analysis of brain homogenates from LMAN1 knockout mice demonstrated that loss of LMAN1 decreases the total protein levels of 5HT 3 A receptors and γ2 subunits of GABA A Rs. LMAN1 knockout regulates the ER proteostasis network by upregulating ERP44 without changing calnexin levels. Interestingly, despite the critical role of the glycanbinding function of LMAN1 in its other known cargo clients, LMAN1 interacts with GABA A Rs in a glycan-independent manner. In summary, LMAN1 is a trafficking factor for certain neuroreceptors in the central nervous system. This is the first report of LMAN1 function in membrane protein trafficking.

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“…In the pHluorin/Myc‐tagged GABA A R pulldown experiment that identified 149 putative GABA A R interactors, IQSEC3 ranked in the middle of the protein list in terms of peptide count 25 . However, IQSEC3 was not identified in a recent GABA A R γ2‐subunit pull down study 5 . Nonetheless, IQSEC3 is clearly involved in the organization of GABAergic synapses, in particular, the correct alignment of the GABAergic pre‐ and postsynaptic compartments 23 .…”

Section: Discussionmentioning

confidence: 56%

“…It consists of multiple GlyRs, each composed of a pentamer of α1 and β subunits clustered by the synaptic scaffolding protein gephyrin (GPHN), and is thought to have limited ability for plastic change 3 . Recent interaction proteomics studies on ligand‐gated ion channels and membrane receptors, such as GABA A and glutamate receptors, have revealed the presence of many protein interactors 4–8 . However, the relatively simple composition known of the GlyR complex (GlyR α1β‐GPHN) may in part be explained by the low sensitive biochemical methods used in the previous studies, leaving additional interactors undetected.…”

Section: Introductionmentioning

confidence: 99%

Glycine Receptor Complex Analysis Using Immunoprecipitation‐Blue Native Gel Electrophoresis‐Mass Spectrometry

et al. 2020

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The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity‐isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β‐GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1β‐GPHN‐IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1β‐GPHN‐IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two‐hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N‐terminal of the IQSEC3 IQ‐like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR‐GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.

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Clptm1 Limits Forward Trafficking of GABAA Receptors to Scale Inhibitory Synaptic Strength

Cited by 63 publications

References 66 publications

Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors

Glycine Receptor Complex Analysis Using Immunoprecipitation‐Blue Native Gel Electrophoresis‐Mass Spectrometry

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