The SLC6/NSS family members KAAT1 and CAATCH1 are weakly Cl--dependent

KAAT1 and CAATCH1, amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta (Castagna et al., 1998, PNAS, 95: 5395-5400; Feldman et al., 2000, J. Biol Chem., 275: 24518-24526), are members of the SLC6/NSS family, which groups neurotransmitter, aminergic transmitter, amino acid and osmolyte transporters coupled to Na+, K+ and Cl- gradients. KAAT1 and CAATCH1 are able to transport different amino acids depending on the contransported cation (Na+ or K+) but their Cl--dependence is not completely defined yet. The Cl- binding site of two members of the family, the serotonin transporter SERT (Forrest et al., 2007, PNAS, 104: 12761-12766) and the GABA transporter GAT1 (Zomot et al., 2007, Nature, 449: 726-730), has been recently modelled on the basis of their functional properties and by structural homology to the leucine transporter LeuT, a prokaryotic member of the family whose three-dimensional structure has been solved at atomic level (Yamashita et al., 2005, Nature, 437: 215-223). The analyses have highlighted the role of a serine residue, that in the Cl--independent LeuT corresponds to Glu 290, and of an asparagine (Asn 286) that also contributes to the coordination of Na+ in the Na1 binding site of LeuT. With the aim to clarify the role exerted by chloride in SLC6/NSS transporters, the Cl--dependence of KAAT1 and CAATCH1 have been here investigated by the expression in Xenopus laevis oocytes and the measurement of induced amino acid uptakes. Despite KAAT1 and CAATCH1 posses the same residue of serine (Ser342, KAAT1 numbering) present in strictly chloride dependent transporters, their transport activities resulted weakly Cl--dependent compared to GAT1. By analysis of the structure of Cl--dependent (SERT and GAT1) and Cl--independent (LeuT) transporters, we selected several residues present in the putative Cl- binding site of KAAT1 and investigated their involvement in chloride interaction.