Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter

Glutamate transporters from the central nervous system play a crucial role in the clearance of the transmitter from the synaptic cleft. Glutamate is c otransported with sodium ions, and the electrogenic translocation cycle is completed by countertransport of potassium. Mutants that cannot interact wi th potassium are only capable of catalyzing electroneutral exchange. Here w e identify a residue involved in controlling substrate recognition in the n euronal transporter EAAC-1 that transports acidic amino acids as well as cy steine, When arginine 447, a residue conserved in all glutamate transporter s, is replaced by cysteine, transport of glutamate or aspartate is abolishe d, but sodium-dependent cysteine transport is left intact. Analysis of othe r substitution mutants shows that the replacement of arginine rather than t he introduced cysteine is responsible for the observed phenotype, In furthe r contrast to wild type, acidic amino acids are unable to inhibit cysteine transport in R447C-EAAC-1, indicating that the selectivity change is manife sted at the binding step, Electrophysiological analysis shows that in the m utant cysteine, transport has become electroneutral, and its interaction wi th the countertransported potassium is impaired. Thus arginine 447 plays a pivotal role in the sequential interaction of acidic amino acids and potass ium with the transporter and, thereby, constitutes one of the molecular det erminants of coupling their fluxes.