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1.
Neuropharmacology ; 238: 109644, 2023 11 01.
Article in English | MEDLINE | ID: mdl-37422181

ABSTRACT

Type-A and -B GABA receptors (GABAARs/GABABRs) control brain function and behaviour by fine tuning neurotransmission. Over-time these receptors have become important therapeutic targets for treating neurodevelopmental and neuropsychiatric disorders. Several positive allosteric modulators (PAMs) of GABARs have reached the clinic and selective targeting of receptor subtypes is crucial. For GABABRs, CGP7930 is a widely used PAM for in vivo studies, but its full pharmacological profile has not yet been established. Here, we reveal that CGP7930 has multiple effects not only on GABABRs but also GABAARs, which for the latter involves potentiation of GABA currents, direct receptor activation, and also inhibition. Furthermore, at higher concentrations, CGP7930 also blocks G protein-coupled inwardly-rectifying K+ (GIRK) channels diminishing GABABR signalling in HEK 293 cells. In male and female rat hippocampal neuron cultures, CGP7930 allosteric effects on GABAARs caused prolonged rise and decay times and reduced the frequency of inhibitory postsynaptic currents and potentiated GABAAR-mediated tonic inhibition. Additional comparison between predominant synaptic- and extrasynaptic-isoforms of GABAAR indicated no evident subtype selectivity for CGP7930. In conclusion, our study of CGP7930 modulation of GABAARs, GABABRs and GIRK channels, indicates this compound is unsuitable for use as a specific GABABR PAM.


Subject(s)
Potassium Channels , Synaptic Transmission , Rats , Male , Humans , Female , Animals , HEK293 Cells , gamma-Aminobutyric Acid , Receptors, GABA-B/metabolism
2.
Sci Signal ; 15(739): eabg2505, 2022 06 21.
Article in English | MEDLINE | ID: mdl-35727864

ABSTRACT

The trans-synaptic adhesion molecule neuroligin-2 (NL2) is essential for the development and function of inhibitory synapses. NL2 recruits the postsynaptic scaffold protein gephyrin, which, in turn, stabilizes γ-aminobutyric acid type A receptors (GABAARs) in the postsynaptic domain. Thus, the amount of NL2 at the synapse can control synaptic GABAAR concentration to tune inhibitory neurotransmission efficacy. Here, using biochemistry, imaging, single-particle tracking, and electrophysiology, we uncovered a key role for cAMP-dependent protein kinase (PKA) in the synaptic stabilization of NL2. We found that PKA-mediated phosphorylation of NL2 at Ser714 caused its dispersal from the synapse and reduced NL2 surface amounts, leading to a loss of synaptic GABAARs. Conversely, enhancing the stability of NL2 at synapses by abolishing PKA-mediated phosphorylation led to increased inhibitory signaling. Thus, PKA plays a key role in regulating NL2 function and GABA-mediated synaptic inhibition.


Subject(s)
Cell Adhesion Molecules, Neuronal , Nerve Tissue Proteins , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phosphorylation , Receptors, GABA-A/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolism
3.
Elife ; 102021 10 06.
Article in English | MEDLINE | ID: mdl-34612814

ABSTRACT

Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes toward LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes.


Subject(s)
A Kinase Anchor Proteins , Calcineurin/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Animals , Escherichia coli , HEK293 Cells , Hippocampus/metabolism , Humans , Long-Term Synaptic Depression , Rats, Sprague-Dawley , Signal Transduction
4.
J Neurosci ; 40(29): 5518-5530, 2020 07 15.
Article in English | MEDLINE | ID: mdl-32513829

ABSTRACT

GABAA receptors (GABAARs) 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 GABAAR gene, GABRA1 This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25 Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with ß3, but not with ß2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential ß subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition.SIGNIFICANCE STATEMENT GABAARs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain, and their dysfunction underlies many neurologic disorders, especially epilepsy. Here we report the characterization of an α1-GABAAR 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 ß subunits resulting in severely reduced synaptic inhibition. Our study reveals that disease-inducing variants can affect GABAAR 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.


Subject(s)
Epilepsy/metabolism , Receptors, GABA-A/biosynthesis , Williams Syndrome/metabolism , Animals , Epilepsy/genetics , Female , HEK293 Cells , Hippocampus/metabolism , Humans , Infant , Male , Neurons/metabolism , Rats, Sprague-Dawley , Receptors, GABA-A/physiology , Williams Syndrome/complications , Williams Syndrome/genetics , Xenopus laevis
5.
Neuropharmacology ; 169: 107540, 2020 06 01.
Article in English | MEDLINE | ID: mdl-30794836

ABSTRACT

GABAA receptors (GABAARs) 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 α1-3ßγ comprise synaptic GABAARs, whilst extrasynaptic α4ßδ, α5ßγ and αß 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 GABAARs 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 GABAAR subunits which mediate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABAARs 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 GABAARs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABAARs are determined by receptor subunit composition and intracellular structural motifs. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.


Subject(s)
Cell Membrane/metabolism , Hippocampus/metabolism , Neurons/metabolism , Receptors, GABA-A/metabolism , Synapses/metabolism , Animals , Cell Membrane/drug effects , Cell Membrane/genetics , Cells, Cultured , Dose-Response Relationship, Drug , HEK293 Cells , Hippocampus/cytology , Hippocampus/drug effects , Humans , Neurons/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/genetics , Synapses/drug effects , Synapses/genetics , gamma-Aminobutyric Acid/pharmacology
6.
J Biol Chem ; 293(35): 13427-13439, 2018 08 31.
Article in English | MEDLINE | ID: mdl-29986886

ABSTRACT

Cell surface expression of type A GABA receptors (GABAARs) is a critical determinant of the efficacy of inhibitory neurotransmission. Pentameric GABAARs are assembled from a large pool of subunits according to precise co-assembly rules that limit the extent of receptor structural diversity. These rules ensure that particular subunits, such as ρ1 and ß3, form functional cell surface ion channels when expressed alone in heterologous systems, whereas other brain-abundant subunits, such as α and γ, are retained within intracellular compartments. Why some of the most abundant GABAAR subunits fail to form homomeric ion channels is unknown. Normally, surface expression of α and γ subunits requires co-assembly with ß subunits via interactions between their N-terminal sequences in the endoplasmic reticulum. Here, using molecular biology, imaging, and electrophysiology with GABAAR chimeras, we have identified two critical residues in the transmembrane domains of α and γ subunits, which, when substituted for their ρ1 counterparts, permit cell surface expression as homomers. Consistent with this, substitution of the ρ1 transmembrane residues for the α subunit equivalents reduced surface expression and altered channel gating, highlighting their importance for GABAAR trafficking and signaling. Although not ligand-gated, the formation of α and γ homomeric ion channels at the cell surface was revealed by incorporating a mutation that imparts the functional signature of spontaneous channel activity. Our study identifies two single transmembrane residues that enable homomeric GABAAR subunit cell surface trafficking and demonstrates that α and γ subunits can form functional ion channels.


Subject(s)
Cell Membrane/metabolism , Receptors, GABA-A/metabolism , Amino Acid Sequence , Animals , Cell Membrane/chemistry , Conserved Sequence , HEK293 Cells , Humans , Mice , Models, Molecular , Protein Domains , Protein Multimerization , Protein Subunits/analysis , Protein Subunits/metabolism , Protein Transport , Receptors, GABA-A/analysis , Signal Transduction , gamma-Aminobutyric Acid/metabolism
7.
Cell Rep ; 23(4): 1060-1071, 2018 04 24.
Article in English | MEDLINE | ID: mdl-29694885

ABSTRACT

The structural and functional plasticity of synapses is critical for learning and memory. Long-term potentiation (LTP) induction promotes spine growth and AMPAR accumulation at excitatory synapses, leading to increased synaptic strength. Glutamate initiates these processes, but the contribution from extracellular modulators is not fully established. Wnts are required for spine formation; however, their impact on activity-mediated spine plasticity and AMPAR localization is unknown. We found that LTP induction rapidly increased synaptic Wnt7a/b protein levels. Acute blockade of endogenous Wnts or loss of postsynaptic Frizzled-7 (Fz7) receptors impaired LTP-mediated synaptic strength, spine growth, and AMPAR localization at synapses. Live imaging of SEP-GluA1 and single-particle tracking revealed that Wnt7a rapidly promoted synaptic AMPAR recruitment and trapping. Wnt7a, through Fz7, induced CaMKII-dependent loss of SynGAP from spines and increased extrasynaptic AMPARs by PKA phosphorylation. We identify a critical role for Wnt-Fz7 signaling in LTP-mediated synaptic accumulation of AMPARs and spine plasticity.


Subject(s)
Long-Term Potentiation/physiology , Neuronal Plasticity/physiology , Receptors, G-Protein-Coupled/metabolism , Receptors, Glutamate/metabolism , Spine/metabolism , Wnt Signaling Pathway/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Frizzled Receptors , Mice , Proto-Oncogene Proteins/metabolism , Spine/cytology , Wnt Proteins/metabolism
8.
Nat Commun ; 8: 14536, 2017 03 06.
Article in English | MEDLINE | ID: mdl-28262662

ABSTRACT

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.


Subject(s)
Hippocampus/metabolism , Microfilament Proteins/genetics , Nerve Tissue Proteins/genetics , Neurons/metabolism , Receptors, GABA-A/genetics , Receptors, GABA-B/genetics , Synaptic Transmission/genetics , Animals , Dentate Gyrus/metabolism , Dentate Gyrus/pathology , Dentate Gyrus/ultrastructure , Embryo, Mammalian , Epilepsy/genetics , Epilepsy/metabolism , Epilepsy/pathology , Gene Expression Regulation , HEK293 Cells , Hippocampus/pathology , Hippocampus/ultrastructure , Humans , Injections, Intraventricular , Intellectual Disability/genetics , Intellectual Disability/metabolism , Intellectual Disability/pathology , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neural Inhibition , Neurogenesis/genetics , Neurons/pathology , Neurons/ultrastructure , Primary Cell Culture , Rats , Rats, Wistar , Receptor Cross-Talk , Receptors, GABA-A/metabolism , Receptors, GABA-B/metabolism , Synapses/metabolism , Synapses/pathology , Synapses/ultrastructure
9.
Cell Rep ; 16(7): 1962-73, 2016 08 16.
Article in English | MEDLINE | ID: mdl-27498877

ABSTRACT

Here, we uncover a mechanism for regulating the number of active presynaptic GABAB receptors (GABABRs) at nerve terminals, an important determinant of neurotransmitter release. We find that GABABRs gain access to axon terminals by lateral diffusion in the membrane. Their relative accumulation is dependent upon agonist activation and the presence of the two distinct sushi domains that are found only in alternatively spliced GABABR1a subunits. Following brief activation of NMDA receptors (NMDARs) using glutamate, GABABR diffusion is reduced, causing accumulation at presynaptic terminals in a Ca(2+)-dependent manner that involves phosphorylation of GABABR2 subunits at Ser783. This signaling cascade indicates how synaptically released glutamate can initiate, via a feedback mechanism, increased levels of presynaptic GABABRs that limit further glutamate release and excitotoxicity.


Subject(s)
Hippocampus/physiology , Presynaptic Terminals/metabolism , Protein Subunits/metabolism , Receptor Cross-Talk , Receptors, GABA-B/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Calcium/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Diffusion , Embryo, Mammalian , Feedback, Physiological , Gene Expression , Glutamic Acid/metabolism , Glutamic Acid/pharmacology , Hippocampus/anatomy & histology , Male , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Phosphorylation , Presynaptic Terminals/drug effects , Protein Subunits/genetics , Rats, Sprague-Dawley , Receptors, GABA-B/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Signal Transduction , Synaptic Transmission , Tissue Culture Techniques , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacology
10.
Stem Cells ; 33(6): 2077-84, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25694335

ABSTRACT

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.


Subject(s)
Aging/physiology , Cell Differentiation/physiology , Down Syndrome/genetics , Induced Pluripotent Stem Cells/cytology , Neurons/cytology , Animals , Cells, Cultured , Fibroblasts/cytology , Humans , Mitochondria/genetics
11.
Neuropharmacology ; 93: 28-40, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25634239

ABSTRACT

The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxß1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2ß1/2γ2 contrasting with the weaker staining of α1/4ß1/2γ2, and no labelling for α3/5/6ß1/2γ2. The labelling of α2ß2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor ß-α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 µM) inhibited GABA-activated currents at all αxß2γ2 receptors examined, but at lower concentrations (5 µM), α-Bgtx was selective for α2ß2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus.


Subject(s)
Brain/cytology , Bungarotoxins/pharmacology , GABA Antagonists/pharmacology , Neurons/drug effects , Animals , Animals, Newborn , Bungarotoxins/metabolism , Embryo, Mammalian , Humans , In Vitro Techniques , Inhibitory Postsynaptic Potentials/drug effects , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred C57BL , Nicotinic Antagonists/pharmacology , Patch-Clamp Techniques , Protein Binding/drug effects , Rats , Receptors, GABA-A/metabolism , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Tubocurarine/pharmacology , gamma-Aminobutyric Acid/pharmacology
12.
Nat Commun ; 5: 4454, 2014 Jul 29.
Article in English | MEDLINE | ID: mdl-25072879

ABSTRACT

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor ß and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation.


Subject(s)
Brain/physiology , GABA Antagonists/metabolism , Receptors, GABA-A/metabolism , Receptors, GABA-A/radiation effects , Synapses/physiology , Ultraviolet Rays , Analysis of Variance , HEK293 Cells , Humans , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Pyridazines , Receptors, GABA-A/genetics
13.
Methods Enzymol ; 521: 109-29, 2013.
Article in English | MEDLINE | ID: mdl-23351736

ABSTRACT

GABA(B) receptors are G-protein-coupled receptors (GPCRs) that are activated by GABA, the principal inhibitory neurotransmitter in the central nervous system. Cell surface mobility of GABA(B) receptors is a key determinant of the efficacy of slow and prolonged synaptic inhibition initiated by GABA. Therefore, experimentally monitoring receptor mobility and how this can be regulated is of primary importance for understanding the roles of GABA(B) receptors in the brain, and how they may be therapeutically exploited. Unusually for a GPCR, heterodimerization between the R1 and R2 subunits is required for the cell surface expression and signaling by prototypical GABA(B) receptors. Here, we describe a minimal epitope-tagging method, based on the incorporation of an α-bungarotoxin binding site (BBS) into the GABA(B) receptor, to study receptor internalization in live cells using a range of imaging approaches. We demonstrate how this technique can be adapted by modifying the BBS to monitor the simultaneous movement of both R1 and R2 subunits, revealing that GABA(B) receptors are internalized as heteromers.


Subject(s)
Bungarotoxins/metabolism , Cell Membrane/metabolism , Cell Tracking/methods , Fluorescent Dyes/analysis , Receptors, GABA-B/analysis , Receptors, GABA-B/metabolism , Animals , Binding Sites , Bungarotoxins/analysis , Cell Membrane/chemistry , Cloning, Molecular/methods , Humans , Models, Molecular , Molecular Imaging/methods , Receptors, GABA-B/genetics , Transfection/methods
14.
Proc Natl Acad Sci U S A ; 109(30): 12171-6, 2012 Jul 24.
Article in English | MEDLINE | ID: mdl-22778417

ABSTRACT

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.


Subject(s)
Hippocampus/metabolism , Protein Subunits/metabolism , Receptors, GABA-B/metabolism , Synaptic Transmission/physiology , Bungarotoxins/metabolism , HEK293 Cells , Hippocampus/physiology , Humans , Image Processing, Computer-Assisted , Patch-Clamp Techniques , Protein Isoforms/genetics , Protein Multimerization , Protein Stability , Protein Structure, Tertiary/physiology , Protein Transport/physiology , Receptors, GABA-B/genetics , Receptors, Metabotropic Glutamate/genetics , Receptors, Metabotropic Glutamate/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
15.
J Biol Chem ; 286(27): 24324-35, 2011 Jul 08.
Article in English | MEDLINE | ID: mdl-21724853

ABSTRACT

γ-Aminobutyric acid type B (GABA(B)) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABA(B) receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABA(B) receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABA(B) receptor.


Subject(s)
Neurons/metabolism , Receptors, GABA-B/metabolism , Signal Transduction/physiology , Synaptic Transmission/physiology , Animals , Bungarotoxins/pharmacology , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , HEK293 Cells , Humans , Protein Multimerization/drug effects , Protein Multimerization/physiology , Protein Stability , Protein Structure, Tertiary , Protein Transport/drug effects , Protein Transport/physiology , Rats , Receptors, GABA-B/genetics , Signal Transduction/drug effects , Synaptic Transmission/drug effects
16.
FEMS Immunol Med Microbiol ; 59(3): 345-9, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20337719

ABSTRACT

We demonstrate that live donor Veillonella dispar cells can transfer the conjugative transposon Tn916 to four different Streptococcus spp. recipients in a multispecies oral consortium growing as a biofilm in a constant depth film fermentor. Additionally, we demonstrate that purified V. dispar DNA can transform Streptococcus mitis to tetracycline resistance in this experimental system. These data show that transfer of conjugative transposon-encoded antibiotic resistance can occur by transformation in addition to conjugation in biofilms.


Subject(s)
Biofilms , Conjugation, Genetic , DNA Transposable Elements , Drug Resistance, Bacterial , Streptococcus mitis/genetics , Transformation, Bacterial , Veillonella/genetics , Anti-Bacterial Agents/pharmacology , DNA, Bacterial/genetics , Humans
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