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1.
Nat Commun ; 14(1): 7511, 2023 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-37980423

RESUMEN

Sodium-dependent glucose transporters (SGLTs) couple a downhill Na+ ion gradient to actively transport sugars. Here, we investigate the impact of the membrane potential on vSGLT structure and function using sugar uptake assays, double electron-electron resonance (DEER), electrostatic calculations, and kinetic modeling. Negative membrane potentials, as present in all cell types, shift the conformational equilibrium of vSGLT towards an outward-facing conformation, leading to increased sugar transport rates. Electrostatic calculations identify gating charge residues responsible for this conformational shift that when mutated reduce galactose transport and eliminate the response of vSGLT to potential. Based on these findings, we propose a comprehensive framework for sugar transport via vSGLT, where the cellular membrane potential facilitates resetting of the transporter after cargo release. This framework holds significance not only for SGLTs but also for other transporters and channels.


Asunto(s)
Simportadores , Simportadores/metabolismo , Azúcares , Glucosa , Potenciales de la Membrana , Galactosa/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Transporte de Sodio-Glucosa/genética , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sodio/metabolismo , Conformación Proteica
2.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-33808202

RESUMEN

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1-5 and TM 6-10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.


Asunto(s)
Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sitios de Unión , Transporte Biológico Activo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glucosa/metabolismo , Lactosa/metabolismo , Modelos Moleculares , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Conformación Proteica , Azúcares/metabolismo , Simportadores/química , Simportadores/metabolismo
3.
Pflugers Arch ; 472(9): 1177-1206, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32767111

RESUMEN

Sodium glucose transporters (SGLTs) belong to the mammalian solute carrier family SLC5. This family includes 12 different members in human that mediate the transport of sugars, vitamins, amino acids, or smaller organic ions such as choline. The SLC5 family belongs to the sodium symporter family (SSS), which encompasses transporters from all kingdoms of life. It furthermore shares similarity to the structural fold of the APC (amino acid-polyamine-organocation) transporter family. Three decades after the first molecular identification of the intestinal Na+-glucose cotransporter SGLT1 by expression cloning, many new discoveries have evolved, from mechanistic analysis to molecular genetics, structural biology, drug discovery, and clinical applications. All of these advances have greatly influenced physiology and medicine. While SGLT1 is essential for fast absorption of glucose and galactose in the intestine, the expression of SGLT2 is largely confined to the early part of the kidney proximal tubules, where it reabsorbs the bulk part of filtered glucose. SGLT2 has been successfully exploited by the pharmaceutical industry to develop effective new drugs for the treatment of diabetic patients. These SGLT2 inhibitors, termed gliflozins, also exhibit favorable nephroprotective effects and likely also cardioprotective effects. In addition, given the recent finding that SGLT2 is also expressed in tumors of pancreas and prostate and in glioblastoma, this opens the door to potential new therapeutic strategies for cancer treatment by specifically targeting SGLT2. Likewise, further discoveries related to the functional association of other SGLTs of the SLC5 family to human pathologies will open the door to potential new therapeutic strategies. We furthermore hope that the herein summarized information about the physiological roles of SGLTs and the therapeutic benefits of the gliflozins will be useful for our readers to better understand the molecular basis of the beneficial effects of these inhibitors, also in the context of the tubuloglomerular feedback (TGF), and the renin-angiotensin system (RAS). The detailed mechanisms underlying the clinical benefits of SGLT2 inhibition by gliflozins still warrant further investigation that may serve as a basis for future drug development.


Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Animales , Descubrimiento de Drogas/métodos , Humanos , Absorción Intestinal , Reabsorción Renal , Proteínas de Transporte de Sodio-Glucosa/antagonistas & inhibidores , Proteínas de Transporte de Sodio-Glucosa/química , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico
4.
Fish Physiol Biochem ; 46(3): 1039-1052, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32062828

RESUMEN

Glucose and fructose play a central role in the metabolism and cellular homeostasis of organisms. Their absorption is co-mediated by two families of glucose transporters, Na+-coupled glucose co-transporters (SGLTs) and facilitative Na+-independent sugar carriers (GLUTs), in the intestine. However, limited information has been available on these transporters in fish. Therefore, we studied glut2, sglt1, and sglt4 genes in grass carp (Ctenopharyngodon idellus). The full-length cDNAs of glut2 was 2308 bp, with an open reading frame (ORF) of 503 amino acids (AAs). The full-length cDNAs of sglt1 was 2890 bp, with an ORF of 658 AAs. Additionally, the full-length cDNAs of sglt4 was 2090 bp, with an ORF encoding 659 AAs. The three deduced AA sequences showed high homology between grass carp and other cyprinid fish species. Based on homology modeling, three-dimensional models of GLUT2, SGLT1, and SGLT4 proteins were created and transmembrane domains were noted. glut2, sglt1, and sglt4 were abundantly expressed in the anterior and mid intestine. In particular, glut2 was markedly expressed in liver (P < 0.05). Additionally, the results indicated that different stocking densities (0.9 or 5.9 kg m-2) did not alter intestinal section-dependent expression patterns of the three transporter genes. However, high stocking density impacted segmental mRNA expression levels. This work demonstrated that mRNA expression of sugar transporter genes in the fish intestine was segment specific, and crowding stress may affect the activity of intestinal sugar transporters. These results provided new insights into the relationship between crowding stress and intestinal sugar transporters in fish.


Asunto(s)
Carpas/genética , Proteínas de Peces/genética , Transportador de Glucosa de Tipo 2/genética , Proteínas de Transporte de Sodio-Glucosa/genética , Secuencia de Aminoácidos , Animales , Acuicultura/métodos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Proteínas de Peces/química , Fructosa , Glucosa , Transportador de Glucosa de Tipo 2/química , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Filogenia , Proteínas de Transporte de Sodio-Glucosa/química
5.
J Mol Model ; 25(7): 186, 2019 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-31187300

RESUMEN

Faced with the worldwide spread of multidrug-resistant (MDR) bacterial strains, together with a lack of any appropriate treatment, urgent steps to combat infectious diseases should be taken. Usually, bacterial components are studied to understand, by analogy, the functioning of human proteins. However, molecular data from bacteria gathered over the past decades provide a sound basis for the search for novel approaches in medical care. With this current work, we want to direct attention to inhibition of the vSGLT glucose transporter from Vibrio parahaemolyticus belonging to the sodium solute symporter (SSS) family, to block sugar transport into the bacterial cell and, as a consequence, to limit its growth. Potential bacteriostatic properties can be drawn from commercially available drugs developed for human diseases. This goal can also be reached with natural components from traditional herbal medicine. The presented data from the numerical analysis of 44 known inhibitors of sodium glucose symporters shed light on potential novel approaches in fighting Gram-negative multidrug-resistant microorganisms. Graphical abstract Molecular view on vSGLT channel inhibition by gneyulin B, the compound of natural origin.


Asunto(s)
Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Proteínas de Transporte de Sodio-Glucosa/química , Estilbenos/química , Antisepsia/métodos , Sitios de Unión , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Ligandos , Unión Proteica , Conformación Proteica , Proteínas de Transporte de Sodio-Glucosa/antagonistas & inhibidores , Estilbenos/farmacología , Vibrio parahaemolyticus/metabolismo
6.
Proc Natl Acad Sci U S A ; 115(12): E2742-E2751, 2018 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-29507231

RESUMEN

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.


Asunto(s)
Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón/métodos , Galactosa/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Micelas , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Conformación Proteica , Sodio/metabolismo , Vibrio parahaemolyticus/química
7.
Mol Phylogenet Evol ; 120: 307-320, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29233707

RESUMEN

Metabolic exchange between cnidarians and their symbiotic dinoflagellates is central to maintaining their mutualistic relationship. Sugars are translocated to the host, while ammonium and nitrate are utilized by the dinoflagellates (Symbiodinium spp.). We investigated membrane protein sequences of each partner to identify potential transporter proteins that move sugars into cnidarian cells and nitrogen products into Symbiodinium cells. We examined the facilitated glucose transporters (GLUT), sodium/glucose cotransporters (SGLT), and aquaporin (AQP) channels in the cnidarian host as mechanisms for sugar uptake, and the ammonium and high-affinity nitrate transporters (AMT and NRT2, respectively) in the algal symbiont as mechanisms for nitrogen uptake. Homologous protein sequences were used for phylogenetic analysis and tertiary structure deductions. In cnidarians, we identified putative glucose transporters of the GLUT family and glycerol transporting AQP proteins, as well as sodium monocarboxylate transporters and sodium myo-inositol cotransporters homologous to SGLT proteins. We hypothesize that cnidarians use GLUT proteins as the primary mechanism for glucose uptake, while glycerol moves into cells by passive diffusion. We also identified putative AMT proteins in several Symbiodinium clades and putative NRT2 proteins only in a single clade. We further observed an upregulation of expressed putative AMT proteins in Symbiodinium, which may have emerged as an adaptation to conditions experienced inside the host cell. This study is the first to identify transporter sequences from a diversity of cnidarian species and Symbiodinium clades, which will be useful for future experimental analyses of the host-symbiont proteome and the nutritional exchange of Symbiodinium cells in hospite.


Asunto(s)
Cnidarios/clasificación , Dinoflagelados/clasificación , Filogenia , Animales , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/clasificación , Proteínas de Transporte de Anión/genética , Acuaporinas/química , Acuaporinas/clasificación , Acuaporinas/genética , Cnidarios/metabolismo , Biología Computacional , Dinoflagelados/metabolismo , Transportadores de Nitrato , Estructura Terciaria de Proteína , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/clasificación , Proteínas de Transporte de Sodio-Glucosa/genética , Simbiosis/fisiología
8.
Protein Sci ; 25(3): 546-58, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26650681

RESUMEN

Glucose is the primary fuel to life on earth. Cellular uptake of glucose is a fundamental process for metabolism, growth, and homeostasis. Three families of secondary glucose transporters have been identified in human, including the major facilitator superfamily glucose facilitators GLUTs, the sodium-driven glucose symporters SGLTs, and the recently identified SWEETs. Structures of representative members or their prokaryotic homologs of all three families were obtained. This review focuses on the recent advances in the structural elucidation of the glucose transporters and the mechanistic insights derived from these structures, including the molecular basis for substrate recognition, alternating access, and stoichiometric coupling of co-transport.


Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Animales , Proteínas Facilitadoras del Transporte de la Glucosa/química , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas de Transporte de Sodio-Glucosa/química , Especificidad por Sustrato
9.
Biochim Biophys Acta ; 1848(11 Pt A): 2799-804, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26260238

RESUMEN

The human sodium-glucose co-transporter 2 (hSGLT2) is a transporter responsible for reabsorption of glucose in the proximal convoluted tubule of the kidney. hSGLT2 inhibitors, including luseogliflozin, have been developed as drugs for type 2 diabetes mellitus. Only luseogliflozin contains a thiosugar ring in its chemical structure, while other hSGLT2 inhibitors contain glucose rings. Consequently, we focused on the binding interactions of hSGLT2 with sugars and thiosugars. We first revealed that the binding affinities of thiosugars are stronger than those of sugars through molecular dynamics simulations of Vibrio parahaemolyticus, sodium-galactose co-transporter, and human hSGLT2. We then demonstrated that Na(+) dissociates from the protein to the cytoplasmic solution more slowly in the thiosugar system than in the sugar system. These differences between sugars and thiosugars are discussed on the basis of the different binding modes due to the atom at the 5-position of the sugar and thiosugar rings. Finally, as a result of Na(+) dissociation, we suggest that the dissociation of thiosugars is slower than that of sugars.


Asunto(s)
Galactosa/química , Glucosa/química , Simulación de Dinámica Molecular , Transportador 2 de Sodio-Glucosa/química , Tioazúcares/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Unión Competitiva , Galactosa/metabolismo , Glucosa/metabolismo , Humanos , Cinética , Estructura Molecular , Análisis de Componente Principal , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sodio/química , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Termodinámica , Tioazúcares/metabolismo , Vibrio parahaemolyticus/metabolismo
10.
J Biol Chem ; 290(1): 127-41, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25398883

RESUMEN

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ~1. In addition, the related and more experimentally tractable SSS member PutP (the Na(+)/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.


Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Proteínas de Escherichia coli/química , Galactosa/química , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/química , Proteínas de Transporte de Sodio-Glucosa/química , Sodio/química , Simportadores/química , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sitios de Unión , Transporte Biológico , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Galactosa/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Sodio/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Homología Estructural de Proteína , Especificidad por Sustrato , Simportadores/metabolismo , Termodinámica , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/metabolismo
11.
PLoS Comput Biol ; 10(12): e1004017, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25522004

RESUMEN

Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11-12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters.


Asunto(s)
Galactosa/química , Galactosa/metabolismo , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Cinética , Simulación de Dinámica Molecular , Sodio/química , Sodio/metabolismo , Termodinámica
13.
Biophys J ; 106(6): 1280-9, 2014 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24655503

RESUMEN

Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 µs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.


Asunto(s)
Simulación de Dinámica Molecular , Proteínas de Transporte de Sodio-Glucosa/química , Algoritmos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Galactosa/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas de Transporte de Sodio-Glucosa/metabolismo
14.
Biochim Biophys Acta ; 1838(1 Pt B): 244-53, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23988430

RESUMEN

The sodium/iodide symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS.


Asunto(s)
Proteínas Bacterianas/química , Yoduros/química , Proteínas de Transporte de Sodio-Glucosa/química , Sodio/química , Simportadores/química , Glándula Tiroides/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Yoduros/metabolismo , Transporte Iónico , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sodio/metabolismo , Proteínas de Transporte de Sodio-Glucosa/genética , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Homología Estructural de Proteína , Simportadores/genética , Simportadores/metabolismo , Glándula Tiroides/metabolismo , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
15.
Proc Natl Acad Sci U S A ; 110(19): 7696-701, 2013 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-23610412

RESUMEN

Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane--a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mechanical coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equilibrium molecular dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.


Asunto(s)
Proteínas de Transporte de Membrana/química , Agua/química , Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Sitios de Unión , Membrana Celular/química , Citoplasma/química , Escherichia coli/química , Humanos , Iones , Simulación de Dinámica Molecular , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/química , Conformación Proteica , Proteínas de Transporte de Sodio-Glucosa/química , Programas Informáticos
16.
Curr Top Membr ; 70: 29-76, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23177983

RESUMEN

Members of the SLC5 and SLC2 family are prominently involved in epithelial sugar transport. SGLT1 (sodium-glucose transporter) and SGLT2, as representatives of the former, mediate sodium-dependent uptake of sugars into intestinal and renal cells. GLUT2 (glucose transporter), as representative of the latter, facilitates the sodium-independent exit of sugars from cells. SGLT has played a major role in the formulation and experimental proof for the existence of sodium cotransport systems. Based on the sequence data and biochemical and biophysical analyses, the role of extramembranous loops in sugar and inhibitor binding can be delineated. Crystal structures and homology modeling of SGLT reveal that the sugar translocation involves operation of two hydrophobic gates and intermediate exofacial and endofacial occluded states of the carrier in an alternating access model. The same basic model is proposed for GLUT1. Studies on GLUT1 have pioneered the isolation of eukaryotic transporters by biochemical methods and the development of transport kinetics and transporter models. For GLUT1, results from extensive mutagenesis, cysteine substitution and accessibility studies can be incorporated into a homology model with a barrel-like structure in which accessibility to the extracellular and intracellular medium is altered by pinching movements of some of the helices. For SGLT1 and GLUT1, the extensive hydrophilic and hydrophobic interactions between sugars and binding sites of the various intramembrane helices occur and lead to different substrate specificities and inhibitor affinities of the two transporters. A complex network of regulatory steps adapts the transport activity to the needs of the body.


Asunto(s)
Células Epiteliales/metabolismo , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Animales , Sitios de Unión , Transporte Biológico Activo , Glucosa/metabolismo , Humanos , Intestino Delgado/metabolismo , Cinética , Modelos Moleculares , Especificidad por Sustrato
17.
Biochim Biophys Acta ; 1818(2): 263-71, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21978597

RESUMEN

Employing molecular dynamics (MD) simulations, the pathway and mechanism of substrate unbinding from the inward-facing state of the Na(+)-coupled galactose transporter, vSGLT, have been investigated. During a 200-ns equilibrium simulation, repeated spontaneous unbinding events of the substrate from its binding site have been observed. In contrast to the previously proposed gating role of a tyrosine residue (Y263), the unbinding mechanism captured in the present equilibrium simulation does not rely on the displacement and/or rotation of this side chain. Rather, the unbinding involves an initial lateral displacement of the substrate out of the binding site which allows the substrate to completely emerge from the region covered by the side chain of Y263 without any noticeable conformational changes of the latter. Starting with the snapshots taken from this equilibrium simulation with the substrate outside the binding site, steered MD (SMD) simulations were then used to probe the translocation of the substrate along the remaining of the release pathway within the protein's lumen and to characterize the nature of protein-substrate interactions involved in the process. Combining the results of the equilibrium and SMD simulations, we provide a description of the full translocation pathway for the substrate release from the binding site into the cytoplasm. Residues E68, N142, T431, and N267 facilitate the initial substrate's displacement out of the binding site, while the translocation of the substrate along the remainder of the exit pathway formed between TM6 and TM8 is facilitated by H-bond interactions between the substrate and a series of conserved, polar residues (Y138, N267, R273, S365, S368, N371, S372, and T375). The observed molecular events indicate that no gating is required for the release of the substrate from the crystallographically captured structure of the inward-facing state of SGLT, suggesting that this conformation might represent an open, rather than occluded, state of the transporter. This article is part of a Special Issue entitled: Membrane protein structure and function.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Galactosa/metabolismo , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sodio/metabolismo , Bacterias/química , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Transporte Biológico , Simulación de Dinámica Molecular , Unión Proteica , Proteínas de Transporte de Sodio-Glucosa/genética
18.
J Biol Chem ; 286(10): 7975-7982, 2011 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-21187287

RESUMEN

SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.


Asunto(s)
Modelos Químicos , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Glucosa , Humanos , Ratones , Mutación Missense , Unión Proteica , Ratas , Proteínas de Transporte de Sodio-Glucosa/genética , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
19.
Biophys J ; 99(7): L56-8, 2010 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-20923633

RESUMEN

It is well accepted that cotransporters facilitate water movement by two independent mechanisms: osmotic flow through a water channel in the protein and flow driven by ion/substrate cotransport. However, the molecular mechanism of transport-linked water flow is controversial. Some researchers believe that it occurs via cotransport, in which water is pumped along with the transported cargo, while others believe that flow is osmotic in response to an increase in intracellular osmolarity. In this letter, we report the results of a 200-ns molecular dynamics simulation of the sodium-dependent galactose cotransporter vSGLT. Our simulation shows that a significant number of water molecules cross the protein through the sugar-binding site in the presence as well as the absence of galactose, and 70-80 water molecules accompany galactose as it moves from the binding site into the intracellular space. During this event, the majority of water molecules in the pathway are unable to diffuse around the galactose, resulting in water in the inner half of the transporter being pushed into the intracellular space and replaced by extracellular water. Thus, our simulation supports the notion that cotransporters act as both passive water channels and active water pumps with the transported substrate acting as a piston to rectify the motion of water.


Asunto(s)
Proteínas Bacterianas/metabolismo , Galactosa/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sodio/metabolismo , Vibrio parahaemolyticus/metabolismo , Agua/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Transporte Biológico , Modelos Moleculares , Ósmosis , Permeabilidad , Proteínas de Transporte de Sodio-Glucosa/química
20.
Artículo en Inglés | MEDLINE | ID: mdl-20570749

RESUMEN

Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut. Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects.


Asunto(s)
Tracto Gastrointestinal/metabolismo , Glucosa/metabolismo , Hemípteros/anatomía & histología , Hemípteros/metabolismo , Proteínas de Insectos/metabolismo , Agua/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Clonación Molecular , ADN Complementario/genética , Femenino , Proteínas Facilitadoras del Transporte de la Glucosa/química , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Hemípteros/efectos de los fármacos , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Absorción Intestinal/efectos de los fármacos , Datos de Secuencia Molecular , Potasio/farmacología , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/genética , Proteínas de Transporte de Sodio-Glucosa/metabolismo
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