Kinetic and pharmacological analysis of L-[35S]-cystine transport into rat brain synaptosomes

Abstract

Abstract The synaptosomal transport of l -[ 35 S]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 ( K m 463±86 μM, V max 185±20 nmol mg protein −1 min −1 ), (b) high affinity sodium-independent transport ( K m 6.90±2.1 μM, V max 0.485±0.060 nmol mg protein −1 min −1 ) and (c) low affinity sodium-independent transport ( K m 327±29 μM, V max 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 -[ 35 S]cystine accumulated into synaptosomes. l -glutamate ( K i 11.2±1.3 μM) was a non-competitive inhibitor of this transporter, and at 100 μM l -glutamate, the V max for l -[ 35 S]cystine transport was reduced to 10% of control. l -cystine did not inhibit the high-affinity sodium-dependent transport of d -[ 3 H]aspartate into synaptosomes. l -histidine and glutathione were the most potent inhibitors of the low affinity sodium-independent transport of l -[ 35 S]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.