Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

Abstract

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 Asp338 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 Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both 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 suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na+ binding site in the recently solved crystal structure of the NSS transporter LeuTAa ( 41 ). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.