Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation

Abstract

Metabotropic GABA B receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA B receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA B receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca 2؉ channels, and removal of extracellular Ca 2؉ prevented glutamate-induced down-regulation of GABA B receptors, indicating that Ca 2؉ influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA B receptors. Glutamate did not affect the rate of GABA B receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA B receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA B receptors by sorting endocytosed GABA B receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA B receptor-mediated inhibition resulting in increased synaptic excitability.

Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid, Type B Receptors

“…Immunocytochemistry and Confocal Laser Scanning Microscopy-Double labeling immunocytochemistry was performed with cortical neurons cultured on coverslips as described previously (10,11,17). Neurons were analyzed by confocal laser scanning microscopy (LSM510 Meta; Zeiss, 100ϫ plan apochromat oil differential interference contrast objective, 1.4 NA) at a resolution of 1,024 ϫ 1,024 pixels in the sequential mode.…”

“…Culture and Transfection of Cortical Neurons-Primary neuronal cultures of cerebral cortex were prepared from day 18 embryos of time-pregnant Wistar rats as described previously (10,11). Neurons were kept in culture for 12-17 days before being used.…”

“…Quantification of fluorescence signals and image processing was done as detailed in Ref. 11. Images shown represent a single optical layer.…”

“…Antibodies-The following primary antibodies were used: rabbit GABA B1a,b (10,11) directed against the C terminus of GABA B1 (affinity-purified, 1:500 for in-cell Western assay and immunofluorescence), rabbit GABA B2N (10,11) directed against the N terminus of GABA B2 (affinity-purified, 1:250 for in-cell Western assay and immunofluorescence, 1:50 for in situ PLA), guinea pig GABA B2 (1:1,000 for immunofluorescence in neurons and 1:4,000 in HEK 293 cells, 1:1,000 for Western blotting; Chemicon International), mouse PDI (1:1,000 for immunofluorescence; Santa Cruz Biotechnology), mouse ubiquitin (P4D1, 1:50 for Western blotting; Santa Cruz Biotechnology), mouse ubiquitin Lys 48 -specific (clone Apu2, 1:50 for in situ PLA; Millipore), mouse VCP (p97) (1:50 for in situ PLA, 3E8DC11; Abcam), mouse actin (1:1,000 for in-cell Western assay; Chemicon International), mouse HA (1:500 for immunofluorescence; Santa Cruz Biotechnology), and rabbit SYVN1/ Hrd1 (1:50 for in situ PLA; Bioss). Secondary antibodies were coupled either to horseradish peroxidase (1:5,000; Jackson ImmunoResearch), Alexa Fluor 488 (1:1,000; Invitrogen), Cy-3 (1:500; Jackson ImmunoResearch), IRDye680 (1:400; LI-COR Biosciences), or IRDye800CW (1:400; LI-COR Biosciences).…”

GABAb Receptor Mediates Opposing Adaptations of GABA Release From Two Types of Prefrontal Interneurons After Observational Fear

“…The possible mechanisms may involve NMDA receptor, which often participates in long-lasting plastic changes and has been shown to control trafficking and surface expression of GABAbR via the CaMKII-AMPK phosphorylation cascade (Guetg et al, 2010;Maier et al, 2010;Terunuma et al, 2010). Changing GABAbR kinetics properties by the KCTD family of axillary proteins (Gassmann and Bettler, 2012;Schwenk et al, 2010) or modulation via several recently discovered interacting proteins (Schwenk et al, 2016) may also be involved.…”