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Regulation of Glial Glutamate Transporters by C-terminal Domains*

A. Leinenweber,Jan-Philipp Machtens,B. Begemann,C. Fahlke

Published 2010 in Journal of Biological Chemistry

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.

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