THE HIGLHY CONSERVED GLYCINE TRIPLET PRESENT IN THE FIRST EXTRACELLULAR LOOP OF KAAT1 IS INVOLVED IN CATION INTERACTION

KAAT1 is a member of the NSS/SLC6 family of solute transporters expressed in the midgut and in salivary glands of Manduca sexta larvae. It realizes the cotransport of neutral amino acids with a peculiar cation selectivity being activated by Na+, Li+ and K+ (Castagna et al., P.N.A.S.: 1998; Ins. Mol. Biol.: 2009; Bossi et al., J. of Physiol.: 1999). As more than 80% of the family members, KAAT1 shows a stretch of three glycines (G85-G87) located at the first extracellular loop (EL1) and according to the crystal structure of LeuT, the protein model of the family (Yamashita et al., Nature: 2005), this glycine triplet is close to the access of the permeation pathway of the transporter. The aim of this work has been investigating the role of this sequence by alanine and cysteine scanning methods and expression in Xenopus laevis oocytes. The surface expression level, measured by a chemilumiscence based approach, was reduced for G85A and G87A mutants but was unaffected for G86A mutant. Nevertheless, all these mutants presented altered uptake activity and transport associated currents in the presence of all the cations that can be exploited by the wt. G85A and G86A mutants in particular showed also reduced uncoupled currents in Na+ and Li+ compared with wt, whereas G87A mutant showed increased uncoupled fluxes in the presence of all the cations. The switch of the entire triplet toward the N terminus of the protein generated a mutant unable to reach the membrane whereas the shift in the opposite direction produced a protein that was localized at the plasma membrane but showed a deep reduction in transport activity. Cross-linking studies performed by the treatment of cysteine mutants with the oxidative complex Cu(II)(1,10-phenanthroline)3 showed that a covalent blockage of position 87 causes a significant reduction of amino acid uptake. The effect was specific and revertible by DTT treatment and, interestingly, Na+ protected G87C mutant from oxidation, probably inducing a movement of the EL1 that increases the distance between the two cysteine partners of the disulfide bond. Our data indicate that the conserved glycine triplet in KAAT1 provides EL1 with the structural flexibility required for the initial steps of cation interaction with the protein.