The Na
+,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in
Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (
Iglu) determined by dual electrode voltage clamp. In
Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed
Iglu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced
Iglu in EAAT3 expressing oocytes. The increased
Iglu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane.
Iglu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant
K127NSGK1 did not significantly alter
Iglu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on
Iglu. The stimulating effect of PIKfyve on
Iglu was abolished by replacement of the serine by alanine in the SGK consensus sequence (
S318APIKfyve). Moreover, additional coexpression of
S318APIKfyve significantly blunted
Iglu in
Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.
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