Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells.
Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.
Hayes
Derek
D
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
Wiessner
Michael
M
Rauen
Thomas
T
McBean
Gethin J
GJ
D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3; D045604:Extracellular Fluid / Q000378:metabolism; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D005978:Glutathione / Q000096:biosynthesis*; D006710:Homocysteine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction / Q000187:drug effects; D027981:Symporters / Q000235:genetics / Q000378:metabolism
D027322:Amino Acid Transport System X-AG; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004151:Dipeptides; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; C493400:SLC1A1 protein, human; C493394:SLC1A3 protein, human; D027981:Symporters; D006710:Homocysteine; C007956:homocysteic acid; C028505:cysteinylglycine; D018698:Glutamic Acid; D003553:Cystine; D005978:Glutathione; D003545:Cysteine