KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K⁺ and Li⁺ in addition to Na⁺. We have previously demonstrated that Asp-338 is essential for KAAT1 activation by K⁺ and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis and expression in Xenopus laevis oocytes, we identified Lys-102 as a residue likely to interact with Asp-338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents both in the presence of Na⁺ and K⁺. However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na⁺-dependent transport and the block in K⁺-dependent transport that characterize the D338E mutant. K⁺-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)3, we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus, both structural and functional evidence suggest that these two residues interact. Similar results have been obtained mutating the bacterial transporter homologue TnaT. Asp-338 corresponds to Asn-286, a residue located in the Na⁺ binding site in the recently solved crystal structure of the NSS transporter LeuT_Aa (41). Our results suggest that Lys-102, interacting with Asp-338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.