Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

Wilkins, Megan E.; Li, Xinyan; Smart, Trevor G.

doi:10.1074/jbc.m803197200

Cited by 54 publications

(59 citation statements)

References 33 publications

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“…1D). These results suggest that as for R1a BBS (21) and R2 BBS (20), inserting a BBS site into R1b enables high affinity BTX binding, and when coexpressed with R2, the pharmacological profile of the receptor is unaffected. Thus, BBS tagging and associated imaging strategies can be used to study the trafficking of R1bR2.…”

Section: Resultsmentioning

confidence: 72%

“…1C), and Scatchard analysis revealed a K d of 32.6 ± 5.1 nM (n = 6). This is eightfold lower than that for α7/5HT 3a receptors (3.9 ± 2.4 nM; n = 3) (20) and threefold lower than BTX binding to R1a BBS in R1aR2 heteromers (9.8 ± 2.6 nM) (21).…”

Section: Resultsmentioning

confidence: 86%

“…The apparent affinity of BTX for its binding site on the GABA B R1b BBS R2 receptor was determined as described previously using [ 125 I]BTX (20,21).…”

Section: Methodsmentioning

confidence: 99%

“…GABA-activated membrane potassium currents were recorded from single transfected GIRK cells using whole-cell patch-clamp recording as indicated previously (21).…”

Section: Methodsmentioning

confidence: 99%

“…Here, we examine how SDs affect GABA B receptor trafficking by inserting the α-bungarotoxin-binding site (BBS) into R1b to distinguish its mobility from R1a subunits. This approach has been successfully applied to monitor the trafficking of R1a and R2 subunits (20,21).…”

mentioning

confidence: 99%

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Sushi domains confer distinct trafficking profiles on GABA _B receptors

Hannan

Wilkins

Smart

2012

Proc. Natl. Acad. Sci. U.S.A.

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GABA B receptors mediate slow inhibitory neurotransmission in the brain and feature during excitatory synaptic plasticity, as well as various neurological conditions. These receptors are obligate heterodimers composed of GABA B R1 and R2 subunits. The two predominant R1 isoforms differ by the presence of two complement control protein modules or Sushi domains (SDs) in the N terminus of R1a. By using live imaging, with an α-bungarotoxin-binding site (BBS) and fluorophore-linked bungarotoxin, we studied how R2 stabilizes R1b subunits at the cell surface. Heterodimerization with R2 reduced the rate of internalization of R1b, compared with R1b homomers. However, R1aR2 heteromers exhibited increased cell surface stability compared with R1bR2 receptors in hippocampal neurons, suggesting that for receptors containing the R1a subunit, the SDs play an additional role in the surface stability of GABA B receptors. Both SDs were necessary to increase the stability of R1aR2 because single deletions caused the receptors to be internalized at the same rate and extent as R1bR2 receptors. Consistent with these findings, a chimera formed from the metabotropic glutamate receptor (mGluR)2 and the SDs from R1a increased the surface stability of mGluR2. These results suggest a role for SDs in stabilizing cell surface receptors that could impart different pre-and postsynaptic trafficking itineraries on GABA B receptors, thereby contributing to their physiological and pathological roles. underlying slow GABA-mediated inhibitory neurotransmission (1) that regulates neuronal excitability (2-4). These receptors are increasingly considered as potential therapeutic targets for a range of diseases, including epilepsy, schizophrenia, anxiety, depression, and substance abuse (1, 5). Functional GABA B receptors are heteromers formed from GABA B R1 subunits containing the agonist binding domain (6) and R2 subunits that link to G protein signaling (7,8).To date, just one isoform of the R2 subunit has been reported (9, 10), whereas several isoforms of the R1 subunit exist [R1a, R1b, R1c, R1e, R1j (1)]. Among these, R1a and R1b predominate in the central nervous system (CNS), and their expression arises by the use of different promoters (11) of the GABBR1 gene. R1a subunits contain 143 aa forming two Sushi domains (SDs) in the N terminus, which are also known as complement control proteins or short consensus repeats (12). These SDs are absent in R1b being replaced by 18 unique amino acids.Heteromers formed from R1aR2 and R1bR2 are thought to play distinct roles in neurotransmission (13-18), with R1aR2 contributing to presynaptic heteroreceptors to inhibit neurotransmitter release, although both R1aR2 and R1bR2 populate postsynaptic membranes to dampen excitability. These heteromeric subtypes exhibit different subcellular compartmentalization with the SDs acting as an axonal-targeting sequence to deliver R1aR2 more efficiently to axons compared with R1bR2 (19). This would, in part, explain the differential pre-and postsynaptic signaling properties o...

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Section: Resultsmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 86%

“…The apparent affinity of BTX for its binding site on the GABA B R1b BBS R2 receptor was determined as described previously using [ 125 I]BTX (20,21).…”

Section: Methodsmentioning

confidence: 99%

“…GABA-activated membrane potassium currents were recorded from single transfected GIRK cells using whole-cell patch-clamp recording as indicated previously (21).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Sushi domains confer distinct trafficking profiles on GABA _B receptors

Hannan

Wilkins

Smart

2012

Proc. Natl. Acad. Sci. U.S.A.

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Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

Thomas

Smart

2012

CP Pharmacology

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Detailed in this unit are protocols for studying the effects of externally and internally applied agents on the behavior of ligand-gated ion channels (LGICs), specifically the GABA(A) receptor. These assays include a number of electrophysiological techniques applied to whole-cell and excised patch recordings of recombinant and native GABA(A) receptor subtypes used in the generation and analysis of a pharmacological data. Although applied to GABA(A) receptors, these techniques are equally applicable to other LGICs. The analysis is extended to incorporate consideration of post-synaptic inhibitory events. In addition, complementary descriptions of how tissues for such studies are prepared for studying recombinant and native receptors are included.

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GABA_B receptors do not internalize after baclofen treatment, possibly due to a lack of β‐arrestin association: Study with a real‐time visualizing assay

Sudo

Hojo

Ando

et al. 2012

Synapse

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The mechanism of agonist-induced GABA(B) receptor (GABA(B) R) internalization is not well understood. To investigate this process, we focused on the interaction of GABA(B) R with β-arrestins, which are key proteins in the internalization of most of the G protein-coupled receptors, and the agonist-induced GABA(B) R internalization and the interaction of GABA(B) R with β-arrestin1 and β-arrestin2 were investigated in real time using GABA(B) R and β-arrestins both of which were fluorescent protein-tagged. We then compared these profiles with those of μ-opioid receptors (μOR), well-studied receptors that associate and cointernalize with β-arrestins. When stimulated by the specific GABA(B) R agonist baclofen, GABA(B) R composed of GABA(B1a) R (GB(1a) R) and fluorescent protein-tagged GABA(B2) R-Venus (GB₂ R-V) formed functional GABA(B) R; they elicited G protein-activated inwardly rectifying potassium channels as well as nontagged GABA(B) R. In cells coexpressing GB(1a) R, GB₂ R-V, and β-arrestin1-Cerulean (βarr1-C) or β-arrestin2-Cerulean (βarr2-C), real-time imaging studies showed that baclofen treatment neither internalized GB₂ R-V nor mobilized βarr1-C or βarr2-C to the cell surface. This happened regardless of the presence of G protein-coupled receptor kinase 4 (GRK4), which forms a complex with GABA(B) R and causes GABA(B) R desensitization. On the other hand, in cells coexpressing μOR-Venus, GRK2, and βarr1-C or βarr2-C, the μOR molecule formed μOR/βarr1 or μOR/βarr2 complexes on the cell surface, which were then internalized into the cytoplasm in a time-dependent manner. Fluorescence resonance energy transfer assay also indicated scarce association of GB₂ R-V and β-arrestins-C with or without the stimulation of baclofen, while robust association of μOR-V with β-arrestins-C was detected after μOR activation. These findings suggest that GABA(B) Rs failure to undergo agonist-induced internalization results in part from its failure to interact with β-arrestins.

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Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

Cited by 54 publications

References 33 publications

Sushi domains confer distinct trafficking profiles on GABA _B receptors

Sushi domains confer distinct trafficking profiles on GABA _B receptors

Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

GABA_B receptors do not internalize after baclofen treatment, possibly due to a lack of β‐arrestin association: Study with a real‐time visualizing assay

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Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

Cited by 54 publications

References 33 publications

Sushi domains confer distinct trafficking profiles on GABA B receptors

Sushi domains confer distinct trafficking profiles on GABA B receptors

Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

GABAB receptors do not internalize after baclofen treatment, possibly due to a lack of β‐arrestin association: Study with a real‐time visualizing assay

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Sushi domains confer distinct trafficking profiles on GABA _B receptors

Sushi domains confer distinct trafficking profiles on GABA _B receptors

GABA_B receptors do not internalize after baclofen treatment, possibly due to a lack of β‐arrestin association: Study with a real‐time visualizing assay