title: The rates of postmortem proteolysis of glutamate transporters differ dramatically between cells and between transporter subtypes.
abstract: Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1-26 and 518-573) had mostly disappeared after 24 hr. GLAST termini (1-25 and 522-543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493-508) and the EAAC1 C-terminus (510-523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493-508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.
authors.affiliation: Department 3 E.N.T., 1st Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
authors.forename: Yuchuan
authors.initials: Y
authors.lastname: Li
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhou
authors.forename: Niels Christian
authors.initials: NC
authors.lastname: Danbolt
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000473:pathology*; D027341:Excitatory Amino Acid Transporter 1 / Q000032:analysis / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000032:analysis / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D011180:Postmortem Changes; D059748:Proteolysis
chemical_list: D027322:Amino Acid Transport System X-AG; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; C493393:Slc1a3 protein, mouse