Functional characterization of a Na+-dependent aspartate transporter from Pyrococcus horikoshii.
Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of GltPh, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described GltPh as an aspartate transporter driven by Na+ or a glutamate transporter driven by either Na+ or H+. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the GltPh transport. We confirm that GltPh is a Na+-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na+ ions. In contrast to the EAATs, transport via GltPh is independent of H+ and K+. We propose a kinetic model of transport in which at least two Na+ ions are coupled to the cotransport of each aspartate molecule by GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.
Ryan
Renae M
RM
Membrane Transport Biophysics Unit, Porter Neuroscience Center, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.
Compton
Emma L R
EL
Mindell
Joseph A
JA
D026905:Amino Acid Transport Systems / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D001692:Biological Transport; D002478:Cells, Cultured; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018698:Glutamic Acid; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D011188:Potassium / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D027341:Excitatory Amino Acid Transporter 1; D008081:Liposomes; D001224:Aspartic Acid; D018698:Glutamic Acid; D006859:Hydrogen; D012964:Sodium; D011188:Potassium