Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2

Abstract

Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na+ ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na+] dependence of ASCT2-associated currents indicates that the Na+/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid–free apo form of the transporter. When the substrate and two Na+ ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na+ ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na+ ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na+ appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.

Na+ Interactions with the Neutral Amino Acid Transporter ASCT1

“…More recently, Zander et al (24) demonstrated that Na ϩ dependence of ASCT2 anion currents is biphasic, suggesting that ASCT2 is coupled to at least two Na ϩ ions. MD simulations in this study similarly suggest the presence of at least two, and possibly three, Na ϩ binding sites in ASCT2 (24). To probe Na ϩ binding sites in ASCTs we mutated coordinating residues within each of the sites proposed for the EAATs and Glt Ph .…”

“…Previous studies have reported a much higher Na ϩ affinity for ASCT2 in the absence of substrate (0.25-2.0 mM) (23,24) compared with the EAATs (EAAT3 is 100 mM (17)). This raises the question: is Na ϩ unbinding during ASCT-mediated exchange, or does Na ϩ function as an allosteric modulator at some of the Na ϩ sites?…”

Structure activity relationships of benzylproline-derived inhibitors of the glutamine transporter ASCT2

“…ASCT2 belongs to the solute carrier 1 (SLC1) family of transporters [3]. It transports neutral amino acids, including glutamine, across the plasma membrane in exchange with an intracellular neutral amino acid [4–5]. The process is dependent on Na + , but not driven by the transmembrane concentration gradient of Na + .…”

Amino Acid Transporters in Drug Discovery

“…This may be valid also for ASCT2, whose rat an human (Fig. 1) homology models have been constructed using the Gltph as template (Albers et al, 2012;Oppedisano et al, 2010;Pingitore et al, 2013;Zander et al, 2013). Significant differences between the rat and humanisoforms, in a region predicted in the vicinity of the substrate binding site were found (Oppedisano et al, 2010).…”

“…The overall transport reaction of hASCT2 follows a random simultaneous mechanism as recently reported in proteoliposomes . A functional aspect still underneath is the electrical nature of the transport reaction; after several contradictory reports (Utsunomiya-Tate et al, 1996;Zander et al, 2013), this issue has been recently dealt with the purified hASCT2, demonstrating a positive regulation by imposed membrane potential due to an electrogenic component in the substrates translocation . Under a physiological point of view, hASCT2 is involved in amino acid absorption in epithelia and other tissues contributing to the glutamine-glutamate cycle in brain and placenta.…”