TY - JOUR T1 - Glutamate Transporter Coupling to Na,K-ATPase JF - The Journal of Neuroscience JO - J. Neurosci. SP - 8143 LP - 8155 DO - 10.1523/JNEUROSCI.1081-09.2009 VL - 29 IS - 25 AU - Erin M. Rose AU - Joseph C. P. Koo AU - Jordan E. Antflick AU - Syed M. Ahmed AU - Stephane Angers AU - David R. Hampson Y1 - 2009/06/24 UR - http://www.jneurosci.org/content/29/25/8143.abstract N2 - 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 α1–α3 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. ER -