The Availability of Surface GABAB Receptors Is Independent of γ-Aminobutyric Acid but Controlled by Glutamate in Central Neurons

Abstract

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of ␥-aminobutyric acid type B (GABA B ) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABA B receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABA B receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABA B receptors endocytose as heterodimers via clathrin-and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABA B receptors in central neurons, consistent with their enrichment at glutamatergic synapses.The efficacy of synaptic transmission depends on the mechanisms of intracellular trafficking, which modify the availability of neurotransmitter receptors (1). At the molecular level, the trafficking of glutamate receptors and ionotropic GABA 5 receptors has been extensively studied (2, 3). For example, differential trafficking of ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors modifies synaptic strength and influences experience-dependent plasticity in vivo (4). However, the molecular mechanisms that govern the trafficking of metabotropic GABA B receptors (GABA B Rs) remain less clear.GABA B Rs mediate the slow component of synaptic inhibition by acting on pre-and postsynaptic targets (5, 6). They have been implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, and cognition (7). They also represent attractive targets for the treatment of withdrawal symptoms from drugs of addiction such as cocaine (8). GABA B Rs are heteromers composed of two subunits, namely GABA B R1 and GABA B R2. GAB-A B R1 contains an endoplasmic reticulum retention motif in the intracellular C-terminal domain (9, 10). The endoplasmic reticulum retention sequence is masked upon assembly with GABA B R2, which results in the appearance of the functional receptor at the plasma membrane. GABA B R1 contains the ligand-binding site, whereas G protein signaling is exclusively mediated by GABA B R2 (11). GABA B Rs are located in dendrites and axons, but it has been difficult to establish what determines their pre-versus postsynaptic localization. Recent evidence suggests that the extracellular domains of the GABA B R1a and -1b isoforms, which differ in two sushi domains (12), may specify axonal/dendritic targeting (13). Unexpectedly, postsynaptic GABA B Rs are enriched at glutamatergic synapses, frequently adjacent to the postsy...

Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABAB receptors in cortical neurons

“…This finding is in line with so far published data obtained under similar experimental conditions irrespective whether receptors were expressed in neurons (Fairfax et al, 2004;Vargas et al, 2008) or in heterologous expression systems (Fairfax et al, 2004;Grampp et al, 2007;Mutneja et al, 2005;Perroy et al, 2003). These experiments suggest that chronic activation does not promote endocytosis of GABA B receptors.…”

“…This conclusion is supported by immunohistochemical data reported on the supraoptic nucleus indicating the presence of GABA B receptors in early endosomes and in lysosomes (Richards et al, 2005). In addition, the presence of endocytosed GABA B receptors in Rab11-positive vesicles was observed using GABA B receptors transiently expressed in spinal cord neurons (Laffray et al, 2007) or hippocampal neurons (Vargas et al 2008). …”

“…On the other hand, colocalization of GABA B receptors with lysosomes as shown in the supraoptic nucleus, argues for a degradation by the classical pathway via lysosomes (Richards et al, 2005). In addition, two recent studies using cultured neurons transfected with epitope-tagged GABA B receptors provided evidence either for constitutive recycling (Vargas et al, 2008) or agonist-induced recycling (Laffray et al, 2007) of the receptors.…”

NMDA receptor-dependent GABA B receptor internalization via CaMKII phosphorylation of serine 867 in GABA B1

“…1 B and C). Upon glutamate treatment (50 μM glutamate/5 μM glycine for 30 min), surface GABA B1b protein was significantly reduced (41.4 ± 5.3% of control, n = 10, P < 0.001), consistent with published data (18). The NMDA receptor antagonist APV (100 μM for 2 h) prevented the glutamate-induced decrease in surface GABA B1b protein (98.4 ± 12.6% of control, n = 9, P > 0.05).…”

“…Activation of GABA B receptors on spines inhibits NMDA receptors through hyperpolarization and the PKA pathway, which enhances Mg 2+ block (13,14) and reduces Ca 2+ permeability (15) of NMDA receptors. Reciprocally, there is evidence that glutamate receptors decrease surface expression of GABA B receptors (16)(17)(18). This supports that glutamate receptors and GABA B receptors cross-talk in dendrites and spines.…”