The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C

Abstract

Treatment of human embryonic kidney 293 cells expressing the rat ~/-aminobutyric acid (GABA) transporter 1 (GATI) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) was found to decrease the velocity of specific [3H]GABA uptake. This downregulation varied with extracellular GABA concentration and was blocked by the PKC inbibitors 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine 017) and staurosporine. An about 50% reduction of uptake velocity by PMA treatment was observed at GABA concentrations > 1 pM, whereas only a minor effect was seen at low substrate concentrations. These data indicate that GATI activity is downregulated by PKC activation.Key words: GABA uptake; Neurotransmitter transporter; Protein kinase C; Rat system, Corey et al. [10] found an upregulation of GABA uptake caused by an increase in the maximal transport velocity (Vmax) upon phorbol ester treatment. This discrepancy may reflect differences in drug application, since the former investigators added the ester to the incubation medium, whereas Corey et al. injected it directly into the oocytes. Here, we used a mammalian cell expression system to re-analyse PKC modulation of this transporter protein. The human embryonic kidney cell line 293 (HEK-293) has been widely employed to express brain membrane proteins [11,12], and we therefore transfected GAT1 cDNA into these cells for investigating the effect of phorbol ester. Our data show that the velocity of GABA uptake mediated by GAT1 is decreased upon activation of PKC. Materials and methods

Second Messengers, Trafficking-Related Proteins, and Amino Acid Residues that Contribute to the Functional Regulation of the Rat Brain GABA Transporter GAT1

“…In addition to the translocation of GAT1 by PMA observed in our studies, independent investigations have shown that, under certain conditions and in different cell types, phorbol esters can exert other effects on GABA transporter function, including: (1) increasing the K m for GABA in oocytes twofold (Osawa et al, 1994); and (2) decreasing V max 50% (at GABA concentrations Ͼ1 M) in 293 cells (Sato et al, 1995). To further understand the mechanisms underlying these multiple effects of phorbol ester treatment and to minimize their impact on the present studies, we examined changes in [ 3 H]GABA uptake and resting membrane potential after different PMA applications.…”

Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases inXenopusoocytes

“…Protein kinase C activation have also been recently shown to modulate the activity of other cloned cotransporters expressed in oocytes and HEK 293 cells [22][23][24][25][26]. The Na/Pi (NaPi-2), Na/Cl/glycine (GLYTlb) cotransporters were inhibited [23,24] and the Na/CI/GABA (GAT1) cotransporter was stimulated after activation of PKC [22].…”

“…3 [22,26]); arachidonic acid reduces the maximal rate of transport by the human Na/glutamate transporter (EAAT1) but stimulates transport by the human EAAT2 isoform [29]. The action of the kinases also depends on the cell type: PKC stimulates the rat GABA transporter expressed in oocytes but inhibits it HEK 293 cells [22,25]. The physiological significance of the differential modulation of cotransporter isoforms by kinases, and the dependence of the kinase action on the cell type are puzzling.…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…GAT1 is also down-regulated after activation of PKC (Osawa et al, 1994;Sato et al, 1995b;Beckman et al, 1998Beckman et al, , 1999Bahena-Trujillo and Arias-Montañ o, 1999;Quick et al, 2004;Wang and Quick, 2005;Cristóvão-Ferreira et al, 2009), but only one study has demonstrated increased transporter phosphorylation upon PKC stimulation (Quick et al, 2004). PKC-mediated Geerlings et al, 2000Geerlings et al, , 2001 N.I., not identified; CIPP, channel-interacting PDZ protein; SNAP-25, synaptosome-associated protein 25; NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2); GPR37, G protein-coupled receptor 37; ARFGAP1, ADP-ribosylation factor GTPase-activating protein 1; Pals1, protein associated with Lin seven 1; Ulip6, Unc-33-like protein 6.…”