Chloride interaction in the SLC6 amino acid cotransporter KAAT1

The SLC6 family of solute transporters groups eukaryotic, Cl--dependent proteins as the serotonin transporter SERT (1) and the GABA transporter GAT1 (2) and prokaryotic, Cl--independent proteins, as the family model LeuT (3, 4). The main role of the anion in the transport cycle appears to be related to the neutralization of the positive charges of sodium ions transported with the substrate. KAAT1 is a lepidopteran SLC6 amino acid cotransporter, activated by Na+ and K+, but characterized by a weak chloride dependence (5, 6). In recent years, taking advantage of the special features of KAAT1, we have investigated the structural/functional relationships within the SLC6 family. By site-directed mutagenesis and functional expression in Xenopus laevis oocytes, we have identified residues involved in Na+ and K+ interaction and in amino acid translocation (7,8,9). The aim of this study has been the identification of the molecular determinants of chloride interaction of KAAT1 to obtain insights in the transport mechanism of SLC6 members. Comparison of KAAT1 sequence with SERT, GAT1 and LeuT has revealed some differences in residues forming the putative anion binding site but, among theme, only T339 seemed to be relevant for chloride dependence: T339S and T339E mutant transport activity became indeed almost completely Cl--dependent. The mutation of a further residue (T67), conserved only in KAAT1 and in the other weakly Cl--dependent transporter of the family CAATCH1, affected KAAT1 activity: T67Y mutant was fully chloride independent whereas T67S and T67A showed an enhancement in chloride dependence. In order to confirm the role of T67, we built the reciprocal mutant of KAAT1 T67Y in GAT1 transporter: Y60T, interestingly, showed a reduced chloride dependence compared to the wt. These data suggest that T67 and T339 influence KAAT1 interaction with chloride. 1. Forrest L.R. et al., Proc Natl Acad Sci U S A. 2007; 104:12761-6. 2. Zomot E. et al., Nature. 2007 Oct 11; 449: 726-30. 3. Yamashita A. et al., Nature. 2005; 437: 215-23 4. Ben-Yona A. et al., J Biol Chem. 2011; 286: 2826-33 5. Castagna M. et al., Proc Natl Acad Sci U S A.; 95: 5395-400 6. Bettè S. et al., Channels (Austin). 2008; 2: 358-62 7. Mari et al., Cell. Mol. Life Sci. 2004, 61: 243-56. 8. Miszner et al., J. Physiol. 2007, 58: 1899-1913. 9. Castagna et al., Am. J. Physiol. Cell. Physiol. 2007, 293: C1286-C1295