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The Journal of Neuroscience, June 24, 2009, 29(25):8143-8155; doi:10.1523/JNEUROSCI.1081-09.2009

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Cellular/Molecular
Glutamate Transporter Coupling to Na,K-ATPase

Erin M. Rose,^1,2 Joseph C. P. Koo,¹ Jordan E. Antflick,¹ Syed M. Ahmed,¹ Stephane Angers,^1,3 and David R. Hampson^1,2

¹Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy and ²Departments of Pharmacology and ³Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3M2, Canada

Correspondence should be addressed to Dr. David R. Hampson, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada. Email: d.hampson{at}utoronto.ca

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 1–3 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 [³H]D-aspartate, [³H]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 µM ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [³H]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.

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Received March 5, 2009; revised May 15, 2009; accepted May 27, 2009.

Correspondence should be addressed to Dr. David R. Hampson, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada. Email: d.hampson{at}utoronto.ca

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