title: Regulation of glial glutamate transporters by C-terminal domains.
abstract: Excitatory amino acid transporter 2 (EAAT2) is a high affinity glutamate transporter predominantly expressed in astroglia. Human EAAT2 encompasses eight transmembrane domains and a 74-amino acid C-terminal domain that resides in the cytoplasm. We examined the role of this region by studying various C-terminal truncations and mutations using heterologous expression in mammalian cells, whole-cell patch clamp recording and confocal imaging. Removal of the complete C terminus (K498X EAAT2) results in loss of function because of intracellular retention of truncated proteins in the cytoplasm. However, a short stretch of amino acids (E500X EAAT2) within the C terminus results in correctly processed transporters. E500X reduced glutamate transport currents by 90%. Moreover, the voltage and substrate dependence of E500X EAAT2 anion currents was significantly altered. WT and mutant EAAT2 anion channels are modified by external Na(+) in the presence as well as in the absence of L-glutamate. Whereas Na(+) stimulates EAAT2 anion currents in the presence of L-glutamate, increased [Na(+)] reduces such currents without glutamate. In cells internally dialyzed with Na(+), WT, and truncated EAAT2 display comparable Na(+) dependence. With K(+) as main internal cation, E500X drastically increased the apparent dissociation constant for external Na(+). The effects of E500X can be represented by a kinetic model that allows translocation of the empty transporter from the outward- to the inward-facing conformation and stabilization of the inward-facing conformation by internal K(+). Our results demonstrate that the C terminus modifies the glutamate uptake cycle, possibly affecting the movements of the translocation domain of EAAT2 glutamate transporter.
authors.affiliation: Institut für Neurophysiologie, Medizinische Hochschule, D-30625 Hannover, Germany.
authors.forename: Ariane
authors.initials: A
authors.lastname: Leinenweber
authors.forename: Jan-Philipp
authors.initials: JP
authors.lastname: Machtens
authors.forename: Birgit
authors.initials: B
authors.lastname: Begemann
authors.forename: Christoph
authors.initials: C
authors.lastname: Fahlke
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D009457:Neuroglia; D011188:Potassium / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins; C493397:SLC1A2 protein, human; D018698:Glutamic Acid; D012964:Sodium; D011188:Potassium