Data di Pubblicazione:
2013
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.
Abstract:
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.
Tipologia IRIS:
14 - Intervento a convegno non pubblicato
Elenco autori:
E. Bossi, M. Giovanola, A. Vollero, F.G. Imperiali, V.F. Sacchi, M. Castagna
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