In this thesis we have investigated some aspects of the molecular physiology of the amino acid transporter KAAT1 and of the GABA transporter GAT1 both belonging to the Neurotransmitter:Sodium Symporter (NSS) family. Wild type as well mutants of these proteins were investigated after expression in Xenopus laevis oocytes and functionally analyzed by radiochemical and electrophysiological assays. In the first chapter a general overview of the biological importance of membrane proteins is provided jointly to a description of the Xenopus oocytes as expression system for this kind of proteins. The second part of the chapter is presenting the general physiological features of KAAT1 and GAT1 while the last part collects information regarding the thermodynamic aspects of secondary active transport as well as the characteristics of the bacterial amino acid transporter LeuT and of the insect dopamine transporter DAT, the two structural models of the NSS family. The last part of the chapter presents one of the most intriguing and cutting edge aspects in the field of membrane transporters: the role of symmetry in the process of ferrying substrates through the lipid bilayer. In chapter 2 are reported the results of our investigation of a highly conserved glycine triplet in KAAT1; this sequence is conserved at the Extracellular Loop 1 (EL1) in almost all the members of the NSS family but the data available at the beginning of our research for GAT1 and for the serotonin transporter SERT were not exhaustive and not in full agreement one to each other. We found that in KAAT1 the flexibility that these amino acids provide to the EL1 is of fundamental importance for the cation access to the extracellular vestibule of the protein. The role that we propose could justify the high degree of conservation showed by this stretch of residues in order to allow the driver ion to get access to its binding site. Different aspects of KAAT1 molecular physiology are addressed in the chapter 3. With our experiments we were able to link the potassium selectivity that characterizes KAAT1 to the polarity of Na1 site and to the dimensional flexibility that is provided in Na2 site by a KAAT1 specific residue of glycine. Beside cation selectivity, we explored the weak chloride dependence of KAAT1 providing new evidences of the fact that its interaction with chloride occurs in an almost unique fashion. The analysis of the 3D homology model of KAAT1 allowed us to identify Thr67 as a residue that we proved experimentally to be a key molecular hinge for the coupling mechanism of ion and substrate flux realized by KAAT1. Furthermore this residue is involved in the initial stereochemical selection that the transporter operates on its substrates as well in the chloride dependence of the transport mechanism. Chapter 4 will briefly resume some preliminary data concerning the possibility to combine the insect Sf9 cell line from Spodoptera frugiperda with the highly efficient Baculovirus expression system with the aim of obtaining the adequate amount of purified protein for KAAT1 crystallization. The last chapter gathers the results of our analysis regarding the influence of internal chloride in the reverse operational mode of the GABA transporter GAT1. Our results provides a link between the oscillations of the intracellular concentration of this anion with the calcium independent GABA release that is described in different pathological and physiological conditions.

MOLECULAR INSIGHTS IN ION ACCESS, DEPENDENCE AND SELECTIVITY IN THE NSS/SLC6 TRANSPORTERS KAAT1 AND GAT1 / M. Giovanola ; tutor: V.F. Sacchi, M. Castagna ; coordinatore: M. Mazzanti. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2014 Jan 21. 26. ciclo, Anno Accademico 2013. [10.13130/giovanola-matteo_phd2014-01-21].

MOLECULAR INSIGHTS IN ION ACCESS, DEPENDENCE AND SELECTIVITY IN THE NSS/SLC6 TRANSPORTERS KAAT1 AND GAT1.

M. Giovanola
2014

Abstract

In this thesis we have investigated some aspects of the molecular physiology of the amino acid transporter KAAT1 and of the GABA transporter GAT1 both belonging to the Neurotransmitter:Sodium Symporter (NSS) family. Wild type as well mutants of these proteins were investigated after expression in Xenopus laevis oocytes and functionally analyzed by radiochemical and electrophysiological assays. In the first chapter a general overview of the biological importance of membrane proteins is provided jointly to a description of the Xenopus oocytes as expression system for this kind of proteins. The second part of the chapter is presenting the general physiological features of KAAT1 and GAT1 while the last part collects information regarding the thermodynamic aspects of secondary active transport as well as the characteristics of the bacterial amino acid transporter LeuT and of the insect dopamine transporter DAT, the two structural models of the NSS family. The last part of the chapter presents one of the most intriguing and cutting edge aspects in the field of membrane transporters: the role of symmetry in the process of ferrying substrates through the lipid bilayer. In chapter 2 are reported the results of our investigation of a highly conserved glycine triplet in KAAT1; this sequence is conserved at the Extracellular Loop 1 (EL1) in almost all the members of the NSS family but the data available at the beginning of our research for GAT1 and for the serotonin transporter SERT were not exhaustive and not in full agreement one to each other. We found that in KAAT1 the flexibility that these amino acids provide to the EL1 is of fundamental importance for the cation access to the extracellular vestibule of the protein. The role that we propose could justify the high degree of conservation showed by this stretch of residues in order to allow the driver ion to get access to its binding site. Different aspects of KAAT1 molecular physiology are addressed in the chapter 3. With our experiments we were able to link the potassium selectivity that characterizes KAAT1 to the polarity of Na1 site and to the dimensional flexibility that is provided in Na2 site by a KAAT1 specific residue of glycine. Beside cation selectivity, we explored the weak chloride dependence of KAAT1 providing new evidences of the fact that its interaction with chloride occurs in an almost unique fashion. The analysis of the 3D homology model of KAAT1 allowed us to identify Thr67 as a residue that we proved experimentally to be a key molecular hinge for the coupling mechanism of ion and substrate flux realized by KAAT1. Furthermore this residue is involved in the initial stereochemical selection that the transporter operates on its substrates as well in the chloride dependence of the transport mechanism. Chapter 4 will briefly resume some preliminary data concerning the possibility to combine the insect Sf9 cell line from Spodoptera frugiperda with the highly efficient Baculovirus expression system with the aim of obtaining the adequate amount of purified protein for KAAT1 crystallization. The last chapter gathers the results of our analysis regarding the influence of internal chloride in the reverse operational mode of the GABA transporter GAT1. Our results provides a link between the oscillations of the intracellular concentration of this anion with the calcium independent GABA release that is described in different pathological and physiological conditions.
21-gen-2014
Settore BIO/09 - Fisiologia
secondary active transporters ; NSS family ; structure-function studies ; glycine rich domains ; ion selectivity ; coupling mechanism ; reverse transport
SACCHI, VELLEA FRANCA
MAZZANTI, MICHELE
Doctoral Thesis
MOLECULAR INSIGHTS IN ION ACCESS, DEPENDENCE AND SELECTIVITY IN THE NSS/SLC6 TRANSPORTERS KAAT1 AND GAT1 / M. Giovanola ; tutor: V.F. Sacchi, M. Castagna ; coordinatore: M. Mazzanti. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2014 Jan 21. 26. ciclo, Anno Accademico 2013. [10.13130/giovanola-matteo_phd2014-01-21].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/232725
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