Kinetic and pharmacological analysis of L-[35S]cystine transport into rat brain synaptosomes.
The synaptosomal transport of L-[35S]cystine occurs by three mechanisms that are distinguishable on the basis of their ionic dependence, kinetics of transport and the specificity of inhibitors. They are (a) low affinity sodium-dependent transport (Km 463 +/- 86 microM, Vmax 185 +/- 20 nmol mg protein-1 min-1), (b) high affinity sodium-independent transport (Km 6.90 +/- 2.1 microM, Vmax 0.485 +/- 0.060 nmol mg protein(-1) min(-1)) and (c) low affinity sodium-independent transport (Km 327 +/- 29 microM, Vmax 4.18 +/- 0.25 nmol mg protein(-1) min(-1)). The sodium-dependent transport of L-cystine was mediated by the X(AG)- family of glutamate transporters, and accounted for almost 90% of the total quantity of L-[35S]cystine accumulated into synaptosomes. L-glutamate (Ki 11.2 +/- 1.3 microM) was a non-competitive inhibitor of this transporter, and at 100 microM L-glutamate, the Vmax for L-[35S]cystine transport was reduced to 10% of control. L-cystine did not inhibit the high-affinity sodium-dependent transport of D-[3H]aspartate into synaptosomes. L-histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of L-[35S]cystine. L-homocysteate, L-cysteine sulphinate and L-homocysteine sulphinate were also effective inhibitors. 1 mM L-glutamate reduced the sodium-independent transport of L-cystine to 63% of control. These results suggest that the vast majority of the L-cystine transported into synaptosomes occurs by the high-affinity glutamate transporters, but that L-cystine may bind to a site that is distinct from that to which L-glutamate binds. The uptake of L-cystine by this mechanism is sensitive to inhibition by increased extracellular concentrations of L-glutamate. The importance of these results for understanding the mechanism of glutamate-mediated neurotoxicity is discussed.
Flynn
J
J
Department of Biochemistry, University College Dublin, Belfield, Ireland.
McBean
G J
GJ
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D003553:Cystine / Q000378:metabolism* / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013574:Synaptosomes / Q000378:metabolism*; D014316:Tritium
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D013462:Sulfur Radioisotopes; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid; D003553:Cystine; D012964:Sodium