The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

Jayanta Mukherjee; Karla Kretschmannova; Geraldine Gouzer; Hans-Michael Maric; Sarah Ramsden; Verena Tretter; Kirsten Harvey; Paul A Davies; Antoine Triller; Hermann Schindelin; Stephen J Moss

doi:10.1523/JNEUROSCI.2001-11.2011

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

Jayanta Mukherjee, Karla Kretschmannova, Geraldine Gouzer, Hans-Michael Maric, Sarah Ramsden, Verena Tretter, Kirsten Harvey, Paul A Davies, Antoine Triller, Hermann Schindelin, Stephen J Moss

112 Citations (Scopus)

Abstract

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.

Original language	English
Journal	The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume	31
Issue number	41
Pages (from-to)	14677-87
Number of pages	11
ISSN	0270-6474
DOIs	https://doi.org/10.1523/JNEUROSCI.2001-11.2011
Publication status	Published - 12 Oct 2011

Keywords

Animals
Calorimetry
Carrier Proteins
Cells, Cultured
DNA-Binding Proteins
Embryo, Mammalian
Female
Hippocampus
Inhibitory Postsynaptic Potentials
Male
Membrane Proteins
Mice
Microscopy, Confocal
Mutation
Neural Inhibition
Neurons
Nuclear Proteins
Patch-Clamp Techniques
Protein Binding
Rats
Receptors, GABA-A
Synapses
Threonine
Transfection
Two-Hybrid System Techniques

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10.1523/JNEUROSCI.2001-11.2011

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Mukherjee, J., Kretschmannova, K., Gouzer, G., Maric, H.-M., Ramsden, S., Tretter, V., Harvey, K., Davies, P. A., Triller, A., Schindelin, H., & Moss, S. J. (2011). The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin. The Journal of neuroscience : the official journal of the Society for Neuroscience, 31(41), 14677-87. https://doi.org/10.1523/JNEUROSCI.2001-11.2011

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin. / Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine et al.
In: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 31, No. 41, 12.10.2011, p. 14677-87.

Research output: Contribution to journal › Journal article › Research › peer-review

Mukherjee, J, Kretschmannova, K, Gouzer, G, Maric, H-M, Ramsden, S, Tretter, V, Harvey, K, Davies, PA, Triller, A, Schindelin, H & Moss, SJ 2011, 'The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 31, no. 41, pp. 14677-87. https://doi.org/10.1523/JNEUROSCI.2001-11.2011

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title = "The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin",

abstract = "The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.",

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author = "Jayanta Mukherjee and Karla Kretschmannova and Geraldine Gouzer and Hans-Michael Maric and Sarah Ramsden and Verena Tretter and Kirsten Harvey and Davies, {Paul A} and Antoine Triller and Hermann Schindelin and Moss, {Stephen J}",

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AU - Mukherjee, Jayanta

AU - Kretschmannova, Karla

AU - Gouzer, Geraldine

AU - Maric, Hans-Michael

AU - Ramsden, Sarah

AU - Tretter, Verena

AU - Harvey, Kirsten

AU - Davies, Paul A

AU - Triller, Antoine

AU - Schindelin, Hermann

AU - Moss, Stephen J

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N2 - The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.

AB - The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABA(A)Rs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABA(A)Rs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABA(A)Rs at synaptic sites, thereby modulating the strength of synaptic inhibition.

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KW - Patch-Clamp Techniques

KW - Protein Binding

KW - Rats

KW - Receptors, GABA-A

KW - Synapses

KW - Threonine

KW - Transfection

KW - Two-Hybrid System Techniques

U2 - 10.1523/JNEUROSCI.2001-11.2011

DO - 10.1523/JNEUROSCI.2001-11.2011

M3 - Journal article

C2 - 21994384

SN - 0270-6474

VL - 31

SP - 14677

EP - 14687

JO - The Journal of neuroscience : the official journal of the Society for Neuroscience

JF - The Journal of neuroscience : the official journal of the Society for Neuroscience

IS - 41

ER -

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

Abstract

Keywords

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