Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody.
Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.
Shashidharan
P
P
Department of Neurology, Mount Sinai School of Medicine, New York, NY 10029, USA. pshashi@smtplink.mssm.edu
Huntley
G W
GW
Murray
J M
JM
Buku
A
A
Moran
T
T
Walsh
M J
MJ
Morrison
J H
JH
Plaitakis
A
A
D000328:Adult; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000032:analysis* / Q000096:biosynthesis; D002460:Cell Line; D002540:Cerebral Cortex / Q000166:cytology*; D002522:Chlorocebus aethiops; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D005971:Glutamates / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013116:Spinal Cord / Q000166:cytology*; D027981:Symporters; D013569:Synapses / Q000648:ultrastructure; D014162:Transfection
D027322:Amino Acid Transport System X-AG; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D005971:Glutamates; D010446:Peptide Fragments; D011994:Recombinant Proteins; C493400:SLC1A1 protein, human; C493399:Slc1a1 protein, mouse; C493398:Slc1a1 protein, rat; D027981:Symporters