• SURFE²R N1

    Easy-to-learn all-in-one device, ideal for teaching and university research
  • SURFE²R N1

    Finally label-free functional assays for transporters available
  • SURFE²R N1

    High signal amplification compared to patch-clamp: transport & binding assays
  • SURFE²R N1

    The only instrument on the market for SSM-based electrophysiology
  • SURFE²R N1

    Turn-key system for efficient transporter protein analysis

2006 - Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii

Icon N1   SURFE²R-technology (custom-built system) publication in Biochemistry (2006)

Authors:
Raunser S., Appel M., Ganea C., Geldmacher-Kaufer U., Fendler K., Kühlbrandt W.

Journal:
Biochemistry (2006) 45(42):12796-805.


Abstract:

The glutamate transporters GltPEc from Escherichia coli and GltPPh from Pyrococcus horikoshii were overexpressed in E. coli and purified to homogeneity with a yield of 1-2 mg/L of culture. Single-particle analysis and electron microscopy indicate that GltP(Ph) is a trimer in detergent solution. Electron microscopy of negatively stained GltPPh two-dimensional crystals shows that the transporter is a trimer also in the membrane. Gel filtration of GltPEc indicates a reversible equilibrium of two oligomeric states in detergent solution that we identified as a trimer and hexamer by blue-native gel electrophoresis and cross-linking. The purified transporters were fully active upon reconstitution into liposomes, as demonstrated by the uptake of radioactively labeled L-aspartate or L-glutamate. L-aspartate/L-glutamate transport of GltPEc involves the cotransport of protons and depends only on pH, whereas GltP(Ph) catalyzes L-glutamate transport with a cotransport of H+ or Na+. L-glutamate induces a fast transient current in GltP(Ph) proteoliposomes coupled to a solid supported membrane (SSM). We show that the electric signal depends on the concentration of Na+ or H+ outside the proteoliposomes and that GltP(Ph) does not require K+ inside the proteoliposomes. In addition, the electrical currents are inhibited by TBOA and HIP-B. The half-saturation concentration for activation of GltPPh glutamate transport (K0.5glut) is 194 µM.


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