Glutamate transporter coupling to Na,K-ATPase.
Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The alpha1-alpha 3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using [(3)H]D-aspartate, [(3)H]L-glutamate, and rubidium-86 uptake into synaptosomes and cultured astrocytes. In synaptosomes, ouabain produced a dose-dependent inhibition of glutamate transporter and Na,K-ATPase activities, whereas in astrocytes, ouabain showed a bimodal effect whereby glutamate transporter activity was stimulated at 1 microm ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [(3)H]D-aspartate uptake indicated the selective involvement of Src kinases, which are probably a component of the Na,K-ATPase/glutamate transporter complex. These findings demonstrate that glutamate transporters and Na,K-ATPases are part of the same macromolecular complexes and operate as a functional unit to regulate glutamatergic neurotransmission.
Rose
Erin M
EM
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada.
Koo
Joseph C P
JC
Antflick
Jordan E
JE
Ahmed
Syed M
SM
Angers
Stephane
S
Hampson
David R
DR
D027322:Amino Acid Transport System X-AG / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D027341:Excitatory Amino Acid Transporter 1 / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*
D027322:Amino Acid Transport System X-AG; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D005904:Glial Fibrillary Acidic Protein; C493392:Slc1a3 protein, rat; D001224:Aspartic Acid; D018698:Glutamic Acid; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium