The crystallization of the bacterial homologue LeuT represented a fundamental step in the comprehension of the molecular physiology of the Solute carrier family 6 (SLC6). From its structure, the macroscopic details of the alternating access model of transport were revealed but the molecular determinants of the coupling mechanism and of the ion selectivity have not been fully elucidated yet; moreover differences are present among the members of the family. One of the main differences is the chloride-dependence, absent in LeuT and in the members of some subfamilies (slc6a20, 19, 15 and 17). However, while in LeuT this independence is explained by the presence of a negatively charge residue in the putative chloride binding site, the members of the cited subfamilies exactly conserve the same sequence of the chloride-dependent members. The insect transporter KAAT1 sits in the middle because of its weak chloride dependence that together with the possibility to utilize K+ as driver ion and the ability of the driver ion to influence the substrate selectivity, unique physiological features, make this transporter a useful tool to investigate the molecular determinants of the coupling mechanism.Comparison with different NSS amino acid sequences and structural analysis point to Threonine 67 in KAAT1 as a possible determinant of the transport coupling mechanism for different reasons: the residue is not conserved among NSS members although it is in a keystone position between the two sodium binding sites of the protein; furthermore, according to the LeuTAa crystal structure, it is involved with its back cone and side chain, in leucine binding. Mutants of T67 were expressed in Xenopus oocytes and functionally characterized by uptake, electrophysiological and chemiluminescence assays. The single point mutation T67Y led to a protein correctly expressed in the oocyte membrane, with unaltered affinity for sodium and with an uptake activity comparable to that of the wild type, but chloride-independent and with a strong reduction of the transport associated current, suggesting the uncoupling of Na+ and amino acid fluxes. The stereoselectivity of the transport process was enhanced as proved by the poor uptake inhibition observed in the presence of the substrate D-leucine. The T67G mutant showed relatively good expression (50-60 % of wt protein - chemiluminescence assay), while its activity was drastically reduced both in uptake and in electrophysiological experiments. These experimental data, together with those reported for GAT1 showing that the reverse mutation (GAT1 Y60T) induced an increased lithium leak current and in sodium dependence, suggest that Thr67 is important in the coupling mechanism, probably participating in the conformational changes that allow the strictly coupled translocation of ion and substrate.
|Titolo:||Residues involved in transport mechanism in the amino acid transporter KAAT1 (SLC6 family)|
|Data di pubblicazione:||2013|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
|Citazione:||Residues involved in transport mechanism in the amino acid transporter KAAT1 (SLC6 family) / E. Bossi, M. Giovanola, A. Vollero, F.G. Imperiali, V.F. Sacchi, M. Castagna. ((Intervento presentato al 6. convegno SFB 35 Symposium - Transmembrane Transporters in Health and Disease tenutosi a Wien nel 2013.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|