Aim: 

KAAT1 is an insect amino acid transporter, member of the NSS/SLC6 family of solute carriers, with peculiar functional properties, being activated by Na⁺ and by K⁺ (Castagna et al., PNAS, 1998). KAAT1 sequence shows a stretch of three consecutive glycines (Gly85 - Gly87) that is highly conserved between members of the family and that, according to topology prediction algorithms and to the crystal structure of the NSS/SLC6 member LeuT (Yamashita et al., Nature, 2005), is located in the extracellular loop 1 (EL1), near the access of permeation pathway.

Methods: 

In the present study we have investigated the functional role of this region by KAAT1 site directed mutagenesis and expression in X. laevis oocytes.

Results: 

The substitution of each glycine with alanine determined a reduction of transport activity of 40 - 50 % compared with wild-type (wt), both in the presence of a Na⁺ or a K⁺ gradient. The shift of the glycine stretch toward the N or the C terminus of the protein, obtained by the synthesis of the double mutants N84G/G87A and G85A/A88G, reduced the activity respectively to 13 and 8 % of wt, whereas the single mutants N84G and A88G showed a residual transport activity that was respectively 40 % and 5 % of wt.

Conclusion: 

Our results indicate that EL1 residues influence KAAT1 activity and that in particular, the conserved three-glycine stretch constitutes a "functional motif" in which not the single glycine residue, but the position of the entire motif plays an essential role in transport function.