Mutations in transmembrane domains 5 and 7 of the human excitatory amino acid transporter 1 affect the substrate-activated anion channel.
L-Glutamate is the predominant excitatory neurotransmitter in the brain, and its extracellular concentration is tightly controlled by the excitatory amino acid transporters (EAATs). The transport of 1 glutamate molecule is coupled to the cotransport of 3 Na+ and 1 H+ and the countertransport of 1 K+. In addition to substrate transport, the binding of glutamate and Na+ activates an anion current which is thermodynamically uncoupled from the transport process. We have identified three amino acid residues in EAAT1 (D272 in TM5, K384 and R385 in TM7) that influence the amplitude of the anion channel current relative to the transport current. Transporters containing the mutations R268A, D272A, D272K, K384A, K384D, R385A, and R385D were expressed in Xenopus laevis oocytes and their transport and anion channel functions measured using the two-electrode voltage clamp techniques. The D272, K384, and R385 mutant transporters showed no change in transport properties but have increased levels of anion channel activity compared to wild-type transporters. These results identify additional residues of the EAAT1 transporter that may contribute to the gating mechanism of the anion channel of glutamate transporters and also provide hints as to how substrate binding leads to channel activation.
Discipline of Pharmacology, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia.
Shiwei
S
Huang
Robert J
RJ
Vandenberg
D019943:Amino Acid Substitution; D000818:Animals; D000838:Anions / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D001693:Biological Transport, Active; D002463:Cell Membrane Permeability; D004594:Electrophysiology; D027341:Excitatory Amino Acid Transporter 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D014982:Xenopus laevis
D000838:Anions; D027341:Excitatory Amino Acid Transporter 1; D007473:Ion Channels; D001224:Aspartic Acid; D018698:Glutamic Acid