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|Authors: ||D'Antoni, Francesca|
|Internal Tutor: ||BOSSI, ELENA|
|Title: ||Properties of the insect neutral amino acis cotransporter KAAT1 studied by alanine scanning mutagenesis and temperature.|
|Abstract: ||The possibility of heterologous expression in Xenopus laevis oocytes has greatly expanded the knowledge of the properties of several cotransporters, membrane proteins that, combining features of pumps and channels, couple the movement of ions and substrates. Transport across the membrane guarantees the reuptake of neurotransmitters from the synaptic cleft, the absorption of nutrients, the regulation of osmolarity and ionic homeostasis. Some of these proteins are involved in neurological diseases and in drug abuse. All the Na+/Cl--dependent cotransporters share high homology and possess the structural topology of the procariotic prototype transporter, LeuT. The present research work was focused on the insect neutral amino acid contransporter KAAT1 (K+-coupled Amino Acid Transporter 1), cloned from the midgut of the larva of Manduca sexta (Castagna et al., 1998;Feldman et al., 2000) and homologous with amino acid transporters belonging to the solute carrier 6 (SLC6) gene family.
The functional role of an highly conserved sequence, consisting of three consecutive glycines (Gly85-Gly86-Gly87), has been investigated by alanine scanning mutagenesis; this sequence is located in the first extracellular loop of the protein, close to Arginine 76, the residue that participates to the external gate of the protein.
The functional characteristics, i.e. transport activity, apparent affinity, interaction with different cations, were derived from electrophysiological measurements (TEVC, Two Electrode Voltage Clamp), and uptake experiments. Additional informations about the correct targeting to the plasma membrane of the glycine mutants have been obtained performing optical measurement of the surface expression by the single oocyte chemiluminescence (SOC) technique pioneered by Zerangue (Zerangue et al., 1999) and used by others (Haerteis et al., 2009;Bossi et al., 2011).
Changes in the transport mechanisms observed for many of the analyzed mutants, indicated a relevant role of the glycine triplet in the transport function, that it was confirmed by the alterations of the uncoupled currents and the apparent affinity.
The second part of my PhD work, covered the analysis of the relationship between temperature and substrate affinity in the ion-coupled cotransporter KAAT1 (from a poikilotherm invertebrate) in comparison with rGAT1, the GABA neuronal transporter originating from a mammalian (homeotherm) animal. Observations based on the rate of transport, and the effects on the presteady-state currents, i.e. the electrical signal arising from the initial steps of the transport cycle (Parent et al., 1992;Parent et al., 1992;Mager et al., 1998;Bossi et al., 1999a) were studied in order to evaluate the kinetic properties of the cotransport system at more physiological temperatures. A strong increase of transmembrane currents was measured in both transporters at higher temperature (30°C); the increased maximal transport-associated current (Imax) was counterbalanced by a lower apparent substrate affinity (K05). The inverse relationship between affinity and current supports previous observations in the intestinal tranporter PepT1(Bossi et al., 2012).|
|Keywords: ||Transporters, structure-function, site-directed mutagenesis, flexibility, temperature, affinity, KAAT1, electrophysiology.|
|Subject MIUR : ||BIO/09 FISIOLOGIA|
|Issue Date: ||2013|
|Doctoral course: ||Fisiologia Sperimentale e Clinica|
|Academic cycle: ||25|
|Publisher: ||Università degli Studi dell'Insubria|
|Citation: ||D'Antoni, F.Properties of the insect neutral amino acis cotransporter KAAT1 studied by alanine scanning mutagenesis and temperature. (Doctoral Thesis, Università degli Studi dell'Insubria, 2013).|
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