The Charge of the Substrate Determines Sodium-Coupling Stoichiometry of the Amino Acid Transporter SLC6A14

SLC6A14 is a member of the SLC6 family of amino acid transporters and is known for its wide selectivity in transporting various amino acids across the cell membrane. A recent report detailed the Na⁺ coupling stoichiometry of SLC6A14 as 3 Na⁺: 1 amino acid substrate, focusing on the transport of the neutral amino acid glycine as the transported substrate. However, it is still unknown how SLC6A14 can also accommodate the transport of amino acids with positively charged side chains. Here, we employed structural modeling and multiple electrophysiological methods to investigate the unique Na⁺/Cl^- coupling mode of SLC6A14. Our results revealed distinct, variable Na⁺ coupling modes when the transporter was subjected to either cationic or neutral amino acids (+, 0). In addition, our data provide further insight into the kinetic mechanism of SLC6A14, demonstrating that positively charged amino acids are transported with a 1.4- to 4-fold slower turnover rate compared to neutral amino acid substrates. We propose a binding mode in which the positively charged amino acid allows binding of and coupling of transport to only two Na⁺ ions, with no effect on Cl^- coupling. Results from molecular dynamics (MD) simulations are consistent with this proposal. These findings have significant implications for our understanding of the substrate selectivity of the transport process as well as the development of new pharmacological compounds targeting this transporter.