Acta Physiologica 2010; Volume 200, Supplement 681
Abstracts of the 61st National Congress of the Italian Physiological Society
DEFINING THE ROLE OF A HIGHLY CONSERVED DOMAIN IN SLC6/NSS FAMILY OF TRANSPORTERS: THE INSECT HOMOLOGUE KAAT1 AS A TOOL
Abstract number: P8
CASTAGNA1 M, SANTACROCE1 M, GIOVANOLA1 M, BOSSI2 E, MARI1 SA, CHERUBINO2 F, SANGALETTI2 R, SACCHI1 VF
1Dept of Molecular Sciences Applied to Biosystems, Univ. degli Studi di Milano, Milan, Italy
2Dept of Biotechnology and Molecular Sciences and Center for Neurosciences, Univ. of Insubria, Varese, Italy
KAAT1 is a transporter characterized by peculiar properties, being activated by Na+, K+and Li+and showing an amino acid selectivity that is influenced by the driver ion. The aim of this work has been investigating the role of a highly conserved triplet of glycines (Gly85 - Gly87), located in the external loop 1, close to the access of the permeation pathway. G85A, G86A and G87A mutants, expressed in X. laevis oocytes, induced altered amino acid uptakes and transport currents in the presence of Na+, Li+and K+: the G85A and G86A showed reduced transport currents and uptakes with all substrates, particularly in leucine, the same was for G87A in leucine, but the mutant behaved like wild type with the other substrates. Investigation of the uncoupled currents showed two interesting aspects: the uncoupled currents were altered, in particular the Li+ current was absent in G85A and reduced in the G86A. The G87A showed instead the greatest currents in all the three tested cations besides the pre-steady state currents were absent. Probably because of the lower expression it was not possible to evaluate this aspect in G85A and G86A. The oxidant Cu(II) (1,10-phenanthroline)3, caused a specific and reversible inhibition of G87C, suggesting that Gly87 is engaged in conformational modifications.
According to the cotransport model for SLC6/NSS family,the results suggest the involvementof the glycine triplet in conformational transitions resulting from the initial interaction with cations.
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Acta Physiologica 2010; Volume 200, Supplement 681 :P8