An anion binding site that regulates the glutamate transporter of synaptic vesicles.
Glutamate, the major excitatory neurotransmitter of the mammalian central nervous system, is stored in synaptic vesicles and released by exocytosis upon depolarization of the presynaptic nerve terminal. Synaptic vesicles possess an active glutamate-specific transporter that is driven by an electrochemical proton gradient across the vesicle membrane and requires chloride for maximal activity. In this study, we have characterized the role of chloride in vesicular glutamate transport using 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent inhibitor of anion translocators. DIDS inhibited glutamate uptake with an IC50 of 0.7 microM or less. In contrast, all energy gradient parameters (membrane potential, pH gradient, and ATPase activity) required at least 5-fold higher concentration of DIDS for inhibition. Furthermore, high concentrations of chloride but not of glutamate or other anions prevented DIDS inhibition of glutamate uptake. In contrast to uptake, glutamate efflux from glutamate-loaded vesicles was independent of chloride over a wide concentration range. However, efflux was still susceptible to DIDS inhibition. DIDS inhibition was prevented by excess chloride. We conclude that the vesicular glutamate transporter possesses a DIDS-sensitive chloride binding site on the cytoplasmic side, distinct from the substrate binding site, which regulates transport activity.
Hartinger
J
J
Howard Hughes Medical Institute, Yale University Medical School, New Haven, Connecticut 06510.
Jahn
R
R
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D027322:Amino Acid Transport System X-AG; D000818:Animals; D000838:Anions; D001665:Binding Sites; D001693:Biological Transport, Active; D002712:Chlorides / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*
D027322:Amino Acid Transport System X-AG; D000838:Anions; D002712:Chlorides; D005971:Glutamates; D006023:Glycoproteins; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid