Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2

ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na + -dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L - cis hydroxyproline biphenyl ester ( Lc -BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc -BPE binding requires one or two Na + ions initially bound to the apo -transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na + ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc -BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc -BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc -BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc -BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.