Neutralization of the aspartic acid residue Asp-367, but not Asp-454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1.
Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na(+) ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na(+) cotransport. To test the effect of charge neutralization mutations in these positions on Na(+) binding to the glutamate-free transporter, we recorded the Na(+)-induced anion leak current to determine the K(m) of EAAC1 for Na(+). For EAAC1(WT), this K(m) was determined as 120 mm. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na(+) activated the anion leak current with a K(m) of about 2 m, indicating dramatically impaired Na(+) binding to the mutant transporter. In contrast, the Na(+) affinity of EAAC1(D454N) was virtually unchanged compared with the wild type transporter (K(m) = 90 mm). The reduced occupancy of the Na(+) binding site of EAAC1(D367N) resulted in a dramatic reduction in glutamate affinity (K(m) = 3.6 mm, 140 mm [Na(+)]), which could be partially overcome by increasing extracellular [Na(+)]. In addition to impairing Na(+) binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na(+) in the glutamate-free transporter form.
Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136, USA.
Zhen
Z
Tao
Zhou
Z
Zhang
Christof
C
Grewer
D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D000838:Anions; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D050580:Excitatory Amino Acid Transporter 3 / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007477:Ions; D007700:Kinetics; D007834:Lasers; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010782:Photolysis; D011485:Protein Binding; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D027322:Amino Acid Transport System X-AG; D000838:Anions; D050580:Excitatory Amino Acid Transporter 3; D007477:Ions; C493400:SLC1A1 protein, human; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium