Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve.
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 SGK1include 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 (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.
Klaus
Fabian
F
Department of Physiology I, University of Tübingen, Germany.
Gehring
Eva-Maria
EM
Zürn
Agathe
A
Laufer
Joerg
J
Lindner
Ricco
R
Strutz-Seebohm
Nathalie
N
Tavaré
Jeremy M
JM
Rothstein
Jeffrey D
JD
Boehmer
Christoph
C
Palmada
Monica
M
Gruner
Ivonne
I
Lang
Undine E
UE
Seebohm
Guiscard
G
Lang
Florian
F
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D050580:Excitatory Amino Acid Transporter 3; D018698:Glutamic Acid; D017874:Immediate-Early Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D000099184:Serum-Glucocorticoid Regulated Kinases; C493400:SLC1A1 protein, human; C492139:PIKFYVE protein, human