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Acta Physiologica Congress

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Acta Physiologica 2011; Volume 203, Supplement 688
The 62nd National Congress of the Italian Physiological Society
9/25/2011-9/27/2011
Sorrento, Italy


THE ROLE OF HIGHLY CONSERVED GLYCINES (8587) IN THE FUNCTION OF THE NEUTRAL AMINO ACID TRANSPORTER KAAT1
Abstract number: P4

DANTONI1 F, GIOVANOLA2 M, SANTACROCE2 M, MARI2 SA, BOSSI1 E, SACCHI2 VF, CASTAGNA2 M

1Dept of Biotechnology and Molecular Sciences and Center for Neurosciences, Univ. of Insubria, Varese, Italy
2Dept of Molecular Sciences Applied to Biosystems, Univ. degli Studi di Milano, Milano, Italy

KAAT1 is a member of the NSS/SLC6 family expressed in the midgut and in salivary glands of Manduca sexta. It performs the transport of neutral amino acids in presence of Na+, Li+ and K+ and has a stretch of three glycines (G85-G87) located at the first extracellular loop (EL1), like many members of the family. According to the crystal structure of LeuT, the bacterial homolog of the family, this glycine triplet is located close to the access of the permeation pathway. The aim of this work was to investigate the role of this highly conserved glycines by alanine and cysteine scanning methods. The wild type (WT) and mutant proteins were expressed in Xenopus laevis oocytes; the surface expression of the G86A mutant was similar to the WT and it was reduced for G85A and G87A. All these mutants had altered uptake activity and transport associated currents; the G85A and G86A mutants in particular showed reduced uncoupled currents in Na+ and Li+ compared with the WT, whereas the G87A mutant showed increased uncoupled fluxes in the presence of all the cations. The shift of the entire triplet toward the N terminus of the protein generated a mutant unable to reach the membrane, while the shift in the opposite direction produced a protein correctly localized in the membrane but with reduced transport activity. Our data indicate that the conserved glycine triplet in KAAT1 provides for the structural flexibility of EL1 that allows the initial steps of transport.

To cite this abstract, please use the following information:
Acta Physiologica 2011; Volume 203, Supplement 688 :P4

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