Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1.
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.
Zhu
Yani
Y
Max-Planck Guest Laboratory at the Institute of Biochemistry and Cell Biology, CAS, 320 Yue Yang Road, Shanghai 200031, China.
Vasilets
Larisa A
LA
Fei
Jian
J
Guo
Lihe
L
Schwarz
Wolfgang
W
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D027322:Amino Acid Transport System X-AG / Q000235:genetics* / Q000502:physiology; D000818:Animals; D000838:Anions; D001120:Arginine; D001692:Biological Transport; D002713:Chlorine / Q000378:metabolism; D004594:Electrophysiology; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009566:Nitrates / Q000378:metabolism; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D027981:Symporters / Q000235:genetics* / Q000502:physiology; D014364:Tryptophan; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D000838:Anions; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D009566:Nitrates; C493399:Slc1a1 protein, mouse; D027981:Symporters; D018698:Glutamic Acid; D002713:Chlorine; D014364:Tryptophan; D001120:Arginine