Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1

Abstract

The excitatory amino acid transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.

Inhibition of Activity of GABA Transporter GAT1 byδ-Opioid Receptor

“…Yolk-free homogenates of oocytes were prepared 3 days after the injection of cRNA as described previously [41] and by passing the oocytes through 200- μ L Eppendorf pipette tips in homogenization buffer (in mM: 20 Tris-HCl (pH 7.4), 5 MgCl 2 , 5 NaH 2 PO 4 , 1 EDTA, 100 NaCl, 10 KCl, 1 DTT, and 1 PMSF, and 5  μ g/mL of each of leupeptin, pepstatin, and antipain). Twenty-microliter aliquots of the yolk-free homogenates were electrophoresed on SDS-PAGE.…”