Regulation of the Glutamate Transporter EAAT4 by PIKfyve

Abstract

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 ^K127NSGK1 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 (^S318APIKfyve). Furthermore, coexpression of ^S318APIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.

Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

“…111 and EAAT4. 150 Whether or not AMPK-sensitivity of PIKfyve contributes to the regulation of those channels and carriers, remains, however, to be shown.…”

PIKfyve Sensitivity of hERG Channels

“…For generation of cRNA [35], constructs were used encoding hERG channels [8], hERG-HA containing an extracellular hemagglutinin epitope [36], human wild type PKB/AKT1 [37,38], wild-type PIKfyve [28], and PKB resistant PIKfyve S318A [28]. The cRNA was generated as described previously [39,40].…”

“…PIKfyve participates in the regulation of retrograde trafficking from the early endosome to the trans-Golgi network (TGN) [22] and thus modifies cell membrane protein trafficking [19,23,24,25,26]. By this means, PIKfyve regulates glucose transporters [11,12,13], creatine transporter [27], glutamate transporters [28,29,30], glutamate receptors [31], Ca 2+ channels [32], Cl - channels [30,33] and inward rectifier potassium channels [34]. …”

Down-Regulation of Na+/K+ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

“…Xenopus oocytes were prepared as previously described [52,53]. cRNA encoding VP1 and VP1(H153A) (10 ng) was injected on the first day after preparation of the Xenopus oocytes [54]. All experiments were performed at room temperature (about 22° C) 3 days after the injection.…”