BIOTINYLATION OF SINGLE CYSTEINE MUTANTS OF THE GLUTAMATE TRANSPORTERGLT-1 FROM RAT-BRAIN REVEALS ITS UNUSUAL TOPOLOGY

In the central nervous system, (Na+ + K+)-coupled glutamate transporte rs restrict the neurotoxicity of this transmitter and limit the durati on of synaptic excitation at some synapses. The Various isotransporter s exhibit a particularly high homology in an extended hydrophobic doma in of ill-defined topology that contains several determinants involved in ion and transmitter binding. Here, we describe the determination o f the membrane topology of the cloned astroglial glutamate transporter GLT-1. A series of functional transporters containing single cysteine s was engineered. Their topological disposition was determined by usin g a biotinylated sulfhydryl reagent. The glutamate transporter has eig ht transmembrane domains long enough to span the membrane as cu helice s. Strikingly, between the seventh and eighth domains, a structure rem iniscent of a pore loop and an outward-facing hydrophobic linker are p ositioned.