Differentiation of substrate and non-substrate inhibitors of transport system xc−: an obligate exchanger of L-glutamate and L-cystine
Introduction
High-affinity glutamate transporters are credited with numerous functions within the mammalian central nervous systems (CNS), including roles in: signal termination, transmitter recycling, excitotoxic protection, and the regulation of extracellular glutamate levels. Many of these activities have been ascribed to the excitatory amino acid transporter (EAAT) subtypes 1–5, which are members of the X−AG family of electrogenic, Na+-dependent anionic amino acid transporters (for review see: Bridges et al., 1999, Danbolt, 2001, Palacin et al., 1998). Another transport system, referred to as system xc−, has been identified which is also capable of translocating L-glutamate, yet is readily distinguished from system X−AG, in that it is Na+-independent, Cl−-dependent, electroneutral, and acts as an obligate exchanger of L-glutamate and L-cystine. Recently cloned from mouse and human cDNA libraries (Bassi et al., 2001, Sato et al., 1999), system xc− is a member of the newly characterized glycoprotein-associated amino acid transporter (gpaAT) family (Verrey et al., 2000). The transporters in this family are all obligate exchangers that must form heterodimers with a glycoprotein subunit (often referred to as the heavy chain) for functional surface expression. In the instance of system xc−, the heterodimer is formed between the transporter subunit xCT, a 502 amino acid containing protein with twelve putative transmembrane domains, and 4F2hc, a cell surface glycoprotein (initially referred to as CD98 in mouse) that is also associated with transport systems L and y+L (Sato et al., 1999).
Rather than function to concentrate L-glutamate intracellularly, this system is thought to utilize normally high intracellular levels of L-glutamate to drive the import of L-cystine. Given this directionality, interest has focused on both the movement of cystine into cells as a precursor for glutathione biosynthesis (Mcbean, 2002, Sagara et al., 1993a), as well as the efflux of glutamate out of cells as a potential route leading to excitotoxic neuronal injury (Barger and Basile, 2001, Espey et al., 1998, Murphy et al., 1989, Piani and Fontana, 1994). As expected, system xc− activity can be quantified using either L-glutamate or L-cystine as a substrate, and in both instances, uptake is Cl−-dependent and Na+-independent. Glutamate uptake through this system is competitively inhibited by cystine and vice versa (Bannai and Kitamura, 1980, Cho and Bannai, 1990). System xc−-mediated transport has been reported to occur in primary cultures of neurons (Sagara et al., 1993b) and astrocytes (Allen et al., 2001, Gochenauer and Robinson, 2001), C6 glioma cells (Cho and Bannai, 1990), human gliomas (Ye et al., 1999), retinal Muller cells (Kato et al., 1993), microglia (Piani and Fontana, 1994), as well as outside the nervous system in fibroblasts, hepatocytes, alveolar type II cells, macrophages, renal tubule cells, and umbilical vein endothelial cells (for review of cellular distributions see Ishii et al., 1992).
The amounts of system xc− activity typically found in many standard neurochemical preparations are relatively low, especially when compared to the levels of EAAT activity. Indeed, the first indications that system xc− was present in brain can be traced back to studies quantifying the distribution and pharmacology of Cl−-dependent L-[3H]glutamate binding, rather than studies directly measuring uptake (Anderson et al., 1990, Bridges et al., 1987a, Bridges et al., 1987b). Advantageously, the identification of CNS-derived tumor cell lines expressing increased levels of Cl−-dependent L- [3H]glutamate uptake allowed the transport system to be more selectively characterized (Murphy et al., 1989, Waniewski and Martin, 1984). The resulting pharmacological profiles from these cell lines are consistent with the activity of system xc− as characterized in non-neural tissue, such as fibroblasts (Bannai and Kitamura, 1980) and hepatocytes (Takada and Bannai, 1984). In the present study we use the rat spinal astrocytoma line originally employed to describe Cl−-dependent glutamate uptake, as well as a more recently characterized human astrocytoma line (i.e., LRM55 and SNB-19 cells, respectively), to further delineate the pharmacological and kinetic properties of system xc−. In addition to identifying a number of competitive inhibitors that block the Cl−-dependent cellular uptake of L-[3H]glutamate, substrate specificity was evaluated with a fluorometric assay that allowed the exchange of extracellular L-cystine with intracellular L-glutamate to be directly followed (Nicholls et al., 1987). In this manner, competitive blockers could be further classified as either alternative substrates, such as ibotenate, or non-substrate inhibitors, such as (S)-4-carboxyphenylglycine. The results of this study should prove of value in constructing a more detailed pharmacological profile of system xc− and identifying analogues with which to investigate its physiological roles, as well as provide more insight into the complex physiological actions of a number of widely utilized glutamate analogues, such as ibotenate and quisqualate, that appear to interact with this transporter.
Section snippets
Chemicals and reagents
General cell culture supplies were purchased from Becton Dickinson (Franklin Lakes, NJ), Corning (Corning, NY), and Life Technologies (Grand Island, NY). L-[3,4-3H]Glutamic acid was purchased from Dupont NEN (Boston, MA). L-Glutamate, L-cystine, L-α-aminoadipate, D-aspartate, dihydrokainate, kainate, L-homocysteate, L-homocysteine sulphinate and L-serine-O-sulphate were purchased from Sigma (St. Louis, MO). (RS)-4-bromohomoibotenate, quisqualate, ibotenate, (S)-4-carboxyphenylglycine, AP4, NMDA
LRM55 cells exhibit marked levels of system xc−-mediated chloride-dependent L-glutamate transport
The uptake of L-[3H]glutamate (100 μM) into LRM55 glioma cells was assessed under various ionic conditions to determine the contribution of Na+-dependent and Cl−-dependent systems to the total observed uptake of L-glutamate in the cell line. Transport in the presence of Cl− and absence of Na+ (equimolar substitution with choline) represented more than 32% (119 ± 7 pmol/min/mg, n = 102) of total uptake in the LRM55 cells measured in the presence of NaCl (374 ± 14 pmol/min/mg, n = 25). Consistent
Discussion
For many years uptake and binding studies with CNS tissue preparations have pointed to the existence of Cl−-dependent glutamate transport systems that are distinct from the prevalent and well-characterized Na+-dependent EAATs (Anderson et al., 1990, Bridges et al., 1986, Bridges et al., 1987b, Kessler et al., 1987, Pin et al., 1984). Only recently, however, has the first of such systems been molecularly characterized (Bassi et al., 2001, Sato et al., 1999). Composed of two distinct protein
Acknowledgements
The authors wish to thank Ben Mickelson for his technical assistance as well as C.M. Thompson, C.S. Esslinger and A.R. Chamberlin for their insightful discussions. This work was supported in part by NINDS NS30570 (RJB) and NCRR RR15583 (RJB, BAW).
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