Central binding sites of LeuT and SERT with a bound molecule of substrate, showing binding sites for Na⁺ that are preserved in all known NSS structures. (A) Outward occluded structure of LeuT (PDB accession no. 2A65) showing a molecule of leucine (yellow) bound at the central site, interacting with Na1 (blue sphere 1) and Y108 (TM3) through its carboxyl group. In addition to the substrate molecule, the Na1 site is formed by side chains from N27 (TM1, T254 [TM6], and N286 [TM7]) as well as main-chain carbonyl oxygen atoms from TM1 and TM6. Na⁺ (blue) at the Na2 site interacts with side chains from T354 and S355 (TM8) and main-chain carbonyls from TM1 and TM8. D404 (TM10) and R30 (TM1) are separated in this structure but form an ion pair in the inward-open LeuT structure (PDB accession no. 3TT3). (B) Outward-occluded model of SERT (Hellsberg et al., 2019) aligned to LeuT in A and showing a molecule of 5-HT (yellow) bound at the central site. The carboxyl group of D98 (TM1) occupies a location corresponding to that of the substrate carboxyl in LeuT and makes similar interactions with Na1 and Y176 (TM3). In addition to D98, side chains from N101 (TM1), S336 (TM6), and N368 (TM7) coordinate the Na⁺ ion. The Na2 site is formed by residues corresponding to those in LeuT. The Cl⁻ ion (green) is bound in SERT at a position corresponding to the carboxyl group of E290 in LeuT and an ion pair is formed between R104 (TM1) and E493 (TM10). Parts of TM6 and TM10 were removed for clarity. TM regions are colored as follows: TM1, salmon; TM3, orange; TM5, lime; TM6, forest; TM7, light teal; TM8, aquamarine; TM10, light blue.