Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

Abstract

Neutral amino acid transporters ASCT1 and ASCT2 are two SLC1 (solute carrier 1) family subtypes, which are specific for neutral amino acids. The other members of the SLC1 family are acidic amino acid transporters (EAATs 1–5). While the functional similarities and differences between the EAATs have been well studied, less is known about how the subtypes ASCT1 and 2 differ in kinetics and function. Here, by performing comprehensive electrophysiological analysis, we identified similarities and differences between these subtypes, as well as novel functional properties, such as apparent substrate affinities of the inward-facing conformation (in the range of 70 μM for L-serine as the substrate). Key findings were: ASCT1 has a higher apparent affinity for Na+, as well as a larger [Na+] dependence of substrate affinity compared to ASCT2. However, the general sequential Na+/substrate binding mechanism with at least one Na+ binding first, followed by amino acid substrate, followed by at least one more Na+ ion, appears to be conserved between the two subtypes. In addition, the first Na+ binding step, presumably to the Na3 site, occurs with high apparent affinity (<1 mM) in both transporters. In addition, ASCT1 and 2 show different substrate selectivities, where ASCT1 does not respond to extracellular glutamine. Finally, in both transporters, we measured rapid, capacitive charge movements upon application and removal of amino acid, due to rearrangement of the translocation equilibrium. This charge movement decays rapidly, with a time constant of 4–5 ms and recovers with a time constant in the 15 ms range after substrate removal. This places a lower limit on the turnover rate of amino acid exchange by these two transporters of 60–80 s−1.

Conserved allosteric inhibition mechanism in SLC1 transporters

“…Therefore, the Na + binding equilibria of the amino acid-free transporter are fully saturated at 140 mM external [Na + ], and the amino acid-induce charge movement can only be caused by Na + binding to the amino acid-bound transporter, substrate translocation, or, most likely, both of those processes. Alanine and serine exchange was previously reported to be rapid in ASCT2 (25). Consistent with this proposal, transient transport current recovered much faster than in EAAT1 after amino acid removal (Fig.…”

“…Amino acid substrate was applied through a theta capillary glass tubing (TG200-4, OD = 2.00 mm, ID = 1.40 mm. Warner Instruments, LLC, MA, USA), with the tip of the theta tubing pulled to a diameter of 350 μm and positioned at 0.5 mm to the cell (25,28). For paired-pulse experiments, currents were recorded with 10/20ms interval time after removal of amino acid.…”

“…HEK293 cells (American Type Culture Collection No. CRL 1573) were cultured as described previously (25). Cell cultures were transiently transfected with wild-type EAAT1 or wild-type/mutant rASCT2 cDNAs, inserted into a modified pBK-CMV-expression plasmid.…”

Do Amino Acid Antiporters Have Asymmetric Substrate Specificity?

“…While the canonical glutamate transporters are symporters, ASCT1 and ASCT2 are obligatory antiporters. Both require Na + for functional antiport [44] but the sodium electrochemical gradient has no apparent effect on the directionality of the fluxes due to high-affinity binding of Na + on both sides of the membrane [45]. Transporters of the SLC1 family form trimers, but do not require any trafficking subunits for surface expression.…”

“…fluxes due to high-affinity of Na + on both sides of the membrane [45]. Transporters of the SLC1 family form trimers, but do not require any trafficking subunits for surface expression.…”