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Scale pubs: = 12 cells). for neuron-specific glutamate transporters in AMPAR synaptic localization and stability. Introduction The glutamatergic system mediates most of the excitatory synaptic activity in the brain and thus plays a critical role in Voxelotor synaptic transmission and higher brain function. After release from the presynaptic terminal, Voxelotor the clearance of glutamate is usually achieved through the coordinated activities of glutamate transporters, i.e., excitatory amino acid transporters (EAATs). To date, three EAATs (EAAT1CEAAT3) have been identified in the hippocampus and cerebral cortex (Pines et al., 1992; Storck et al., 1992; Arriza et al., 1994; Rothstein et al., 1994). The glial transporters GLAST/EAAT1 and GLT/EAAT2 are primarily localized to the plasma membrane of astrocytic processes that wrap the synapse (Rothstein et al., 1994; Chaudhry et al., 1995). In contrast, EAAC1/EAAT3 is usually expressed in the presynaptic and postsynaptic regions of neurons (Rothstein et al., 1994; He et al., 2000, 2001). Although glial transporters conduct the majority of glutamate reuptake (Rothstein et al., 1996; Peghini et al., 1997; Tanaka et al., 1997), a number of studies suggest that neuronal glutamate transport may play an important role in the dynamics of transmitter clearance (Diamond, 2001; Scimemi et al., 2009). Indeed, in the hippocampal CA1 region, more than half of the synapses do not have an astrocytic process in close proximity, suggesting that neuronal glutamate transport may play a larger role than originally thought Rabbit polyclonal to NOTCH1 in confining released glutamate (Ventura and Harris, 1999). Consistently, EAAT3 knockdown reveals reduced glutamate uptake, neurodegeneration, and epilepsy (Rothstein et al., 1996). It has been well established that AMPA receptor (AMPAR) trafficking and redistribution serve as fundamental mechanisms for synaptic plasticity and higher brain functions, including learning and memory, but how EAAT function affects glutamate receptors remains unclear. Conceivably, the three key components Voxelotor of the glutamatergic systemthe agonist glutamate and its two binding partners (receptors and transporters)may be functionally coordinated. AMPARs are highly dynamic, trafficking constantly between the plasma membrane and intracellular compartments in a glutamate-dependent manner. The levels of cell-surface AMPARs are regulated by the rate Voxelotor of receptor insertion and internalization, whereas the total amount of AMPARs is determined by a balance between synthesis and degradation. Given that EAAT function is usually closely coupled with synaptic activity, and AMPAR dynamics and turnover are sensitive to neural activation, EAAT activity may play a role in the regulation of AMPARs. Here we report that this synaptic localization and protein stability of AMPARs are regulated by glutamate transporter activity. Suppression of EAAT results in AMPAR endocytosis, leading to a reduction in AMPAR synaptic and surface localization. After internalization that is likely initiated by AMPAR ubiquitination, AMPARs are sorted to the proteasome for degradation. We find that this EAAT effect on AMPAR trafficking and degradation requires activation of the parasynaptically distributed NR2B-containing NMDARs, indicating a role of glutamate spillover during transporter inhibition. Furthermore, inhibition of glial EAAT shows no effect on AMPAR levels, whereas selective knockdown of neuronal glutamate transporter EAAT3, specifically Voxelotor those localized at the postsynaptic domain name, induces a reduction in AMPAR expression. These results indicate the presence of functional crosstalk between glutamate transporters and receptors at synaptic sites. Materials and Methods Antibodies and reagents. Antibodies and reagents were obtained from the following commercial sources: anti-GluA1nt (N-terminal), anti-GluA1ct (C-terminal), anti-GluA2nt (N-terminal), and anti-GluN1 were from Millipore; anti-tubulin, anti-pan-p38 MAPK, ifenprodil, -methyl-4-carboxyphenylglycine (MCPG), z-leu-leu-leu-al (MG132), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dihydrokainic acid (DHK), ammonium chloride, chloroquine, lactacystin, dimethylsulfoxide (DMSO), and 5-fluoro-2-deoxyuridine (FDU) were from Sigma-Aldrich; dl-threo–benzyloxyaspartic acid (TBOA), ()-threo-3-methylglutamic acid (T3MG), APV, and MK801 were from Tocris Bioscience; and anti-phospho-p38 and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580, SB) were from Cell Signaling Technology. Anti-ubiquitin (P4D1) was obtained from Santa Cruz Biotechnology,.