Hippocampal glutamate transporter 1 (GLT-1) complex levels are paralleling memory training in the Multiple T-maze in C57BL/6J mice.

ABSTRACT

The glutamate transporter 1 (GLT-1) is essential for glutamate uptake in the brain and associated with various psychiatric and neurological disorders. Pharmacological inhibition of GLT-1 results in memory deficits, but no study linking native GLT-1 complexes was published so far. It was therefore the aim of the study to associate this highly hydrophobic, eight transmembrane spanning domains containing transporter to memory training in the Multiple T-maze (MTM). C57BL/6J mice were used for the spatial memory training experiments, and trained mice were compared to untrained (yoked) animals. Mouse hippocampi were dissected out 6 h after training on day 4, and a total enriched membrane fraction was prepared by ultracentrifugation. Membrane proteins were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) with subsequent Western blotting against GLT-1 on these native gels. Moreover, GLT-1 complexes were identified by mass spectrometry (nano-LC-ESI-MS/MS). Animals learned the MTM task and multiple GLT-1 complexes were detected at apparent molecular weights of 242, 480 and 720 kDa on BN-PAGE Western blotting. GLT-1 complex levels were significantly higher in the trained group as compared to yoked controls, and antibody specificity was verified by immunoblotting on multidimensional gels. Hippocampal GLT-1 was unambiguously identified by mass spectrometry with high sequence coverage, and glycosylation was observed. It is revealed that increased GLT-1 complex levels are paralleling and are linked to spatial memory training. We provide evidence that signal termination, represented by the excitatory amino acid transporter GLT-1 complexes, is involved in spatial memory mechanisms.