Alesutan Ioana S, Ureche Oana N, Laufer Joerg, Klaus Fabian, Zürn Agathe, Lindner Ricco, Strutz-Seebohm Nathalie, Tavaré Jeremy M, Boehmer Christoph, Palmada Monica, Lang Undine E, Seebohm Guiscard, Lang Florian
Department of Physiology I, University of Tübingen, D-72076 Tübingen, Germany.
Cell Physiol Biochem. 2010;25(2-3):187-94. doi: 10.1159/000276569. Epub 2010 Jan 12.
The excitatory amino-acid transporter EAAT4 (SLC1A6), a Na(+),glutamate cotransporter expressed mainly in Purkinje cells, serves to clear glutamate from the synaptic cleft. EAAT4 activity is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent regulation of the Na(+),glucose transporter SGLT1 (SLC5A1) and the creatine transporter CreaT (SLC6A8) has recently been shown to involve the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether SGK1-dependent EAAT4-regulation similarly involves PIKfyve. In Xenopus oocytes expressing EAAT4, but not in water injected oocytes, glutamate induced a current which was significantly enhanced by coexpression of PIKfyve and SGK1. The glutamate induced current in Xenopus oocytes coexpressing EAAT4 and both, PIKfyve and SGK1, was significantly larger than the current in Xenopus oocytes expressing EAAT4 together with either kinase alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter glutamate induced current in EAAT4-expressing Xenopus oocytes and abolished the stimulation of glutamate induced current by coexpression of PIKfyve. The stimulating effect of PIKfyve was abrogated by replacement of the serine with alanine in the SGK consensus sequence ((S318A)PIKfyve). Furthermore, coexpression of (S318A)PIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.
兴奋性氨基酸转运体EAAT4（溶质载体家族1成员6，SLC1A6）是一种主要在浦肯野细胞中表达的钠依赖性谷氨酸协同转运体，其作用是清除突触间隙中的谷氨酸。血清和糖皮质激素诱导激酶SGK1可刺激EAAT4的活性。最近研究表明，SGK1对钠依赖性葡萄糖转运体SGLT1（溶质载体家族5成员1，SLC5A1）和肌酸转运体CreaT（溶质载体家族6成员8，SLC6A8）的调节作用涉及哺乳动物磷脂酰肌醇-3-磷酸-5-激酶PIKfyve（磷脂酰肌醇磷酸激酶3，PIP5K3）。因此，本实验探讨了SGK1对EAAT4的调节作用是否同样涉及PIKfyve。在表达EAAT4的非洲爪蟾卵母细胞中，而非注射水的卵母细胞中，谷氨酸可诱导产生电流，共表达PIKfyve和SGK1可显著增强该电流。在共表达EAAT4、PIKfyve和SGK1的非洲爪蟾卵母细胞中，谷氨酸诱导产生的电流明显大于共表达EAAT4与任一激酶的非洲爪蟾卵母细胞中的电流。共表达无活性的SGK1突变体（K127N）SGK1不会显著改变表达EAAT4的非洲爪蟾卵母细胞中谷氨酸诱导产生的电流，且可消除共表达PIKfyve对谷氨酸诱导电流的刺激作用。在SGK共有序列中将丝氨酸替换为丙氨酸（（S318A）PIKfyve）可消除PIKfyve的刺激作用。此外，共表达（S318A）PIKfyve可显著减弱SGK1对EAAT4活性的刺激作用。这些观察结果表明，PIKfyve确实参与了对EAAT4的调节。
