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Structure-function studies on a sodium-coupled phosphate cotransporter


Patti, Monica. Structure-function studies on a sodium-coupled phosphate cotransporter. 2013, University of Zurich, Faculty of Science.

Abstract

Type II sodium-phosphate cotransporters (NaPi-II), encoded by the SLC34 gene family, play an important physiological role in the homeostasis of inorganic phosphate (Pi). Three protein isoforms are found in mammals designated NaPi-IIa, NaPi-IIb and NaPi-IIc (or SLC34A1,2,3). They are integral membrane proteins that mediate uphill Pi transport driven by the electrochemical gradient of sodium. Their tissue localization determines their physiological role: whereas NaPi-IIa and NaPi-IIc are mainly expressed in the renal proximal tubule, NaPi-IIb is expressed in the small intestine and other non-renal epithelial-like cells. Disturbed phosphate homeostasis is associated with many diseases. The ability to modulate the activity of NaPi-II in controlling phosphate absorption could have a large clinical impact, particularly in end stage renal disease where the ability to excrete phosphate is compromised, leading to high plasma levels and vascular calcification. For this reason the investigation of the structure-function relationships of these transport proteins has clinical relevance for the development of specifically targeted drugs. Three themes are covered in this thesis: Characterization of the cotransport dynamics Protein conformational changes occurring during the transport cycle can be investigated with voltage clamp fluorometry (VCF), whereby a thiol reactive fluorescence molecule is used as an indirect reporter of motion. This technique applied to NaPi-IIb containing substituted cysteines has revealed complementary movement of two protein domains during the transport cycle. Using VCF applied to mutant transporters that retain their basic function after labelling with the fluorophore, a more detailed “map” of the movements of different domains of the protein during the transport cycle under physiological conditions was obtained. Moreover, by combining VCF and presteady-state analysis, we investigated the correlation of kinetics of voltage-dependent transitions (manifested as presteady-state relaxations) with the kinetics of localized microenvironment changes, reported by changes in fluorescence intensity (ΔF). A direct correlation would suggest that the fluorophore senses the interaction of ions and the charge movement attributed to the empty carrier. Our findings allow us to relate protein rearrangements with the voltage-dependent events during the transport cycle such as substrate binding and translocation and predict the location of cation binding sites. Identification of an internal cation release step Knowledge of the internal release and binding of substrates to NaPi-II is limited. New insight was obtained using the electroneutral NaPi-IIc isoform. Electrogenicity of NaPi-IIc was restored by replacing of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc. The Na+:Pi stoichiometry of this engineered electrogenic construct (AAD-IIc) increased from 2:1 to 3:1. Accompanying this fundamental functional change, AAD-IIc also showed a different behaviour from the wild-type electrogenic NaPi-IIa/b, specifically a reduced apparent Pi affinity and different presteady-state kinetics. This compromised behaviour was investigated using electrophysiology and voltage clamp fluorometry (VCF). The activation energy of AAD-IIc was considerably different from NaPi-IIc and NaPi-IIa; in particular AAD-IIc shows a higher activation energy associated with the empty carrier reorientation. These studies have shown that the AAD-IIc retains the electroneutral cooperative interaction of 2 Na+ ions of the electroneutral NaPi-IIc and the presteady-state charge relaxations of AAD-IIc are mainly due to the empty carrier (in the absence of external Pi), and to the cytosolic release of one Na+ ion (in the presence of Pi). Using simulations of presteady-state and steady-state behavior, it was possible to identify two critical partial reactions: the final release of Na+ to the cytosol and external Pi binding. Moreover, using VCF with Cys-mutants of AAD-IIc we confirmed the predictions from the model simulation. Homology model and substrate binding sites localization The transport mechanism and structure-function relationships of sodium-dependent phosphate cotransporter (NaPi-II) have been extensively studied; however, its 3D structure is still unknown. Although the structures of some sodium-dependent cotransporters have been resolved, the classical bioinformatic approaches fail to identify a suitable template for the creation of homology model of NaPi-II. Using hydrophobicity profiles and hidden Markov Models it was possible to define a structural repeat common to all NaPi-II isoforms. Now, using the recently solved crystal structure of Vibrio cholerae Na+-dicarboxylate transporter VcINDY as a template, we were able to generate a homology model of human NaPi-IIa. The predicted Na+ and Pi coordination sites are localized in specific repeat motifs in the center of the protein and furthermore biochemical and electrophysiological investigations have confirmed the importance of key amino acids predicted to be involved in substrate coordination.

Abstract

Type II sodium-phosphate cotransporters (NaPi-II), encoded by the SLC34 gene family, play an important physiological role in the homeostasis of inorganic phosphate (Pi). Three protein isoforms are found in mammals designated NaPi-IIa, NaPi-IIb and NaPi-IIc (or SLC34A1,2,3). They are integral membrane proteins that mediate uphill Pi transport driven by the electrochemical gradient of sodium. Their tissue localization determines their physiological role: whereas NaPi-IIa and NaPi-IIc are mainly expressed in the renal proximal tubule, NaPi-IIb is expressed in the small intestine and other non-renal epithelial-like cells. Disturbed phosphate homeostasis is associated with many diseases. The ability to modulate the activity of NaPi-II in controlling phosphate absorption could have a large clinical impact, particularly in end stage renal disease where the ability to excrete phosphate is compromised, leading to high plasma levels and vascular calcification. For this reason the investigation of the structure-function relationships of these transport proteins has clinical relevance for the development of specifically targeted drugs. Three themes are covered in this thesis: Characterization of the cotransport dynamics Protein conformational changes occurring during the transport cycle can be investigated with voltage clamp fluorometry (VCF), whereby a thiol reactive fluorescence molecule is used as an indirect reporter of motion. This technique applied to NaPi-IIb containing substituted cysteines has revealed complementary movement of two protein domains during the transport cycle. Using VCF applied to mutant transporters that retain their basic function after labelling with the fluorophore, a more detailed “map” of the movements of different domains of the protein during the transport cycle under physiological conditions was obtained. Moreover, by combining VCF and presteady-state analysis, we investigated the correlation of kinetics of voltage-dependent transitions (manifested as presteady-state relaxations) with the kinetics of localized microenvironment changes, reported by changes in fluorescence intensity (ΔF). A direct correlation would suggest that the fluorophore senses the interaction of ions and the charge movement attributed to the empty carrier. Our findings allow us to relate protein rearrangements with the voltage-dependent events during the transport cycle such as substrate binding and translocation and predict the location of cation binding sites. Identification of an internal cation release step Knowledge of the internal release and binding of substrates to NaPi-II is limited. New insight was obtained using the electroneutral NaPi-IIc isoform. Electrogenicity of NaPi-IIc was restored by replacing of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc. The Na+:Pi stoichiometry of this engineered electrogenic construct (AAD-IIc) increased from 2:1 to 3:1. Accompanying this fundamental functional change, AAD-IIc also showed a different behaviour from the wild-type electrogenic NaPi-IIa/b, specifically a reduced apparent Pi affinity and different presteady-state kinetics. This compromised behaviour was investigated using electrophysiology and voltage clamp fluorometry (VCF). The activation energy of AAD-IIc was considerably different from NaPi-IIc and NaPi-IIa; in particular AAD-IIc shows a higher activation energy associated with the empty carrier reorientation. These studies have shown that the AAD-IIc retains the electroneutral cooperative interaction of 2 Na+ ions of the electroneutral NaPi-IIc and the presteady-state charge relaxations of AAD-IIc are mainly due to the empty carrier (in the absence of external Pi), and to the cytosolic release of one Na+ ion (in the presence of Pi). Using simulations of presteady-state and steady-state behavior, it was possible to identify two critical partial reactions: the final release of Na+ to the cytosol and external Pi binding. Moreover, using VCF with Cys-mutants of AAD-IIc we confirmed the predictions from the model simulation. Homology model and substrate binding sites localization The transport mechanism and structure-function relationships of sodium-dependent phosphate cotransporter (NaPi-II) have been extensively studied; however, its 3D structure is still unknown. Although the structures of some sodium-dependent cotransporters have been resolved, the classical bioinformatic approaches fail to identify a suitable template for the creation of homology model of NaPi-II. Using hydrophobicity profiles and hidden Markov Models it was possible to define a structural repeat common to all NaPi-II isoforms. Now, using the recently solved crystal structure of Vibrio cholerae Na+-dicarboxylate transporter VcINDY as a template, we were able to generate a homology model of human NaPi-IIa. The predicted Na+ and Pi coordination sites are localized in specific repeat motifs in the center of the protein and furthermore biochemical and electrophysiological investigations have confirmed the importance of key amino acids predicted to be involved in substrate coordination.

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Other titles:Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich
Item Type:Dissertation
Referees:Verrey F, Forster I C, Wagner C A, Kellenberger Stephan
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2013
Deposited On:07 Nov 2013 12:36
Last Modified:05 Apr 2016 17:07
Number of Pages:143

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