Functional Role of a Highly Conserved Sequence Motif in the Insect Amino Acid Transporter KAAT1

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+ (1). KAAT1 sequence shows a stretch of three consecutive glycines (Gly85 - Gly87) that is highly conserved between members of the family (2, 3) and that, according to topology prediction algorithms and to the crystal structure of the NSS/SLC6 member LeuT (4) is located in the extracellular loop 1 (EL1), near the access of the permeation pathway. In the present study we have investigated the functional role of this region by KAAT1 site directed mutagenesis and expression in X. laevis oocytes. The substitution of each glycine with alanine determined a reduction of transport activity of 40 - 50 % compared with wild-type (wt) (S.E. of 5%, n = 56). 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% ± 1 (n = 25) and 8% ± 2 (n = 27) of wt, whereas the single mutants N84G and A88G showed a residual transport activity that was respectively 36 % ± 6 (n =31) and 5 % ± 1 (n = 38) of wt. 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. 1.Castagna et al., (1998), PNAS, 95: 5395-5400 2.Zhou and Kanner, (2005) J.Biol.Chem.280: 20316-20324. 3.Mao et al., (2008) Mol. Memb. Biol. 25: 115-127. 4.Yamashita et al., (2005) Nature. 437: 215-223