期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 34, 页码 14297-14302出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0904625106
关键词
cation binding site; obligate exchange mutant; sodium selectivity
资金
- National Institute of Neurological Disorders and Stroke (NINDS)
- National Institutes of Health [NS16708]
- U.S.-Israel Binational Science Foundation [2007051]
Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. Their transport cycle consists of two separate translocation steps, namely cotransport of glutamic acid with three Na+ followed by countertransport of K+. Two Tl+ binding sites, presumed to serve as sodium sites, were observed in the crystal structure of a related archeal homolog and the side chain of a conserved aspartate residue contributed to one of these sites. We have mutated the corresponding residue of the eukaryotic glutamate transporters GLT-1 and EAAC1 to asparagine, serine, and cysteine. Remarkably, these mutants exhibited significant sodium-dependent radioactive acidic amino acid uptake when expressed in HeLa cells. Reconstitution experiments revealed that net uptake by the mutants in K+-loaded liposomes was impaired. However, with Na+ and unlabeled L-aspartate inside the liposomes, exchange levels were around 50-90% of those by wild-type. In further contrast to wild-type, where either substrate or K+ stimulated the anion conductance by the transporter, substrate but not K+ modulated the anion conductance of the mutants expressed in oocytes. Both with wild-type EAAC1 and EAAC1-D455N, not only sodium but also lithium could support radioactive acidic amino acid uptake. In contrast, with D455S and D455C, radioactive uptake was only observed in the presence of sodium. Thus the conserved aspartate is required for transporter-cation interactions in each of the two separate translocation steps and likely participates in an overlapping sodium and potassium binding site.
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