RESUMEN
In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.
Asunto(s)
Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Dominio Catalítico/fisiología , Fluorometría/métodos , Técnicas de Placa-Clamp/métodos , Transportador 1 de Sodio-Glucosa/química , Transportador 1 de Sodio-Glucosa/metabolismo , Animales , Sitios de Unión/genética , Dominio Catalítico/efectos de los fármacos , Cisteína , Expresión Génica , Glucosa/metabolismo , Iones/metabolismo , Modelos Animales , Modelos Moleculares , Técnicas de Sonda Molecular , Mutación , Oocitos/metabolismo , Polietilenglicoles/química , Conformación Proteica , Rodaminas/farmacología , Sodio/metabolismo , Transportador 1 de Sodio-Glucosa/genética , Simportadores/metabolismo , Xenopus laevisRESUMEN
Membrane transporters, in addition to their major role as specific carriers for ions and small molecules, can also behave as water channels. However, neither the location of the water pathway in the protein nor their functional importance is known. Here, we map the pathway for water and urea through the intestinal sodium/glucose cotransporter SGLT1. Molecular dynamics simulations using the atomic structure of the bacterial transporter vSGLT suggest that water permeates the same path as Na+ and sugar. On a structural model of SGLT1, based on the homology structure of vSGLT, we identified and mutated residues lining the sugar transport pathway to cysteine. The mutants were expressed in Xenopus oocytes, and the unitary water and urea permeabilities were determined before and after modifying the cysteine side chain with reversible methanethiosulfonate reagents. The results demonstrate that water and urea follow the sugar transport pathway through SGLT1. The changes in permeability, increases or decreases, with side-chain modifications depend on the location of the mutation in the region of external or internal gates, or the sugar binding site. These changes in permeability are hypothesized to be due to alterations in steric hindrance to water and urea, and/or changes in protein folding caused by mismatching of side chains in the water pathway. Water permeation through SGLT1 and other transporters bears directly on the structural mechanism for the transport of polar solutes through these proteins. Finally, in vitro experiments on mouse small intestine show that SGLT1 accounts for two-thirds of the passive water flow across the gut.
Asunto(s)
Proteínas de Transporte de Membrana/fisiología , Transportador 1 de Sodio-Glucosa/metabolismo , Agua/química , Agua/metabolismo , Animales , Acuaporinas/metabolismo , Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Mucosa Intestinal/metabolismo , Transporte Iónico/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Simulación de Dinámica Molecular , Mutación , Oocitos/metabolismo , Presión Osmótica , Permeabilidad , Sodio/metabolismo , Transportador 1 de Sodio-Glucosa/química , Urea/metabolismo , Xenopus/metabolismoRESUMEN
SGLT2 inhibitors are a new class of drugs that have been recently developed to treat type II diabetes. They lower glucose levels by inhibiting the renal Na(+)/glucose cotransporter SGLT2, thereby increasing the amount of glucose excreted in the urine. Pharmacodynamics studies have raised questions about how these inhibitors reach SGLT2 in the brush border membrane of the S1 and S2 segments of the renal proximal tubule: are these drugs filtered by the glomerulus and act extracellularly, or do they enter the cell and act intracellularly? To address this question we expressed hSGLT2 in HEK-293T cells and determined the affinity of a specific hSGLT2 inhibitor, TA-3404 (also known as JNJ-30980924), from the extra- and intracellular side of the plasma membrane. Inhibition of SGLT2 activity (Na(+)/glucose currents) by TA-3404 was determined using the whole-cell patch clamp that allows controlling the composition of both the extracellular and intracellular solutions. We compared the results to those obtained using the nonselective SGLT inhibitor phlorizin, and to the effect of TA-3404 on hSGLT1. Our results showed that TA-3404 is a potent extracellular inhibitor of glucose inward SGLT2 transport (IC50 2 nmol/L) but it was ineffective from the intracellular compartment at both low (5 mmol/L) and high (150 mmol/L) intracellular NaCl concentrations. We conclude that TA-3404 only inhibits SGLT2 from the extracellular side of the plasma membrane, suggesting that it is filtered from the blood through the glomerulus and acts from within the tubule lumen.
RESUMEN
Sodium glucose cotransporters (SGLTs) mediate the translocation of carbohydrates across the brush border membrane of different organs such as intestine, kidney, and brain. The human SGLT5 (hSGLT5), in particular, is localized in the kidney were it is responsible for mannose and fructose reabsorption from the glomerular filtrate as confirmed by more recent studies on hSGLT5 knockout mice. Here we characterize the functional properties of hSGLT5 expressed in a stable T-Rex-HEK-293 cell line using biochemical and electrophysiological assays. We confirmed that hSGLT5 is a sodium/mannose transporter that is blocked by phlorizin. Li(+) and H(+) ions were also able to drive mannose transport, and transport was electrogenic. Our results moreover indicate that substrates require a pyranose ring with an axial hydroxyl group (-OH) on carbon 2 (C-2). Compared with Na(+)/glucose cotransport, the level of function of Na(+)/mannose cotransport in rat kidney slices was low.
Asunto(s)
Riñón/metabolismo , Manosa/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sodio/metabolismo , Animales , Cationes , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Riñón/efectos de los fármacos , Cinética , Litio/metabolismo , Potenciales de la Membrana , Estructura Molecular , Florizina/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte de Sodio-Glucosa/antagonistas & inhibidores , Proteínas de Transporte de Sodio-Glucosa/genética , TransfecciónRESUMEN
Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na(+) in binding sites is beyond the resolution of the structures, two Na(+) binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na(+) binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na(+) and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na(+) binding, and that Na(+) binding to Na2 promotes binding to Na1 and also sugar binding.
Asunto(s)
Sitios de Unión/genética , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Transportador 1 de Sodio-Glucosa/metabolismo , Sodio/metabolismo , Biofisica/métodos , Humanos , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Técnicas de Placa-Clamp , Unión Proteica , Transportador 1 de Sodio-Glucosa/genéticaRESUMEN
SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.
Asunto(s)
Variación Genética/genética , Proteínas de Transporte de Membrana/genética , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Perros , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Proteínas de Transporte de Membrana/fisiología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Prolina/genética , Unión Proteica/genética , Transporte de Proteínas/genética , Serina/genética , Sodio/deficienciaRESUMEN
Human concentrative nucleoside transporter 3 (hCNT3) uses the electrochemical gradient of Na(+) and H(+) to drive the transport of nucleosides and therapeutic nucleoside analogs into the cells. We employed the two-electrode voltage clamp technique to compare the steady-state and presteady-state kinetics of hCNT3 in the presence of Na(+) and H(+). We found that H(+) supported a higher maximal rate of uridine transport than Na(+), but the efficiency of transport was lower. For both cations, maximal uridine-induced current increased with hyperpolarizing potentials and did not saturate within the voltage range tested. Apparent affinity of hCNT3 for uridine in H(+) was insensitive to membrane voltage at negative potentials, and decreased with depolarization. In contrast, apparent affinity for uridine in Na(+) decreased with hyperpolarization and was independent of voltage at depolarizing potentials. H(+)-coupled hCNT3 exhibited lower affinity for all natural nucleosides and different substrate selectivity compared to Na(+)-coupled hCNT3. In H(+), lack of the hydroxyl groups at 2' and 5' decreased the affinity, while lack of the nitrogen N-7 or inversion of the configuration of the hydroxyl group at 2' prevented transport. Presteady-state charge movements of hCNT3 did not decrease when extracellular cation concentration (Na(+) or H(+)) was reduced, but the tau(ON)-V and Q-V curves were shifted to more negative potentials. The different effects of uridine and inosine on presteady-state currents in H(+) indicated a change in rate-limiting step for the transport of these substrates by H(+)-coupled hCNT3.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Protones , Sodio/metabolismo , Uridina/metabolismo , Adenosina/metabolismo , Animales , Femenino , Humanos , Cinética , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
The Na(+)-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K(0.5)) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I(max)) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I(max). Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I(max). Fludarabine and formycin B were also transported with higher I(max) than uridine and moderate affinity (102 +/- 10 and 66 +/- 6 microM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. K(0.5) for Na(+) was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na(+) per nucleoside. This discrepancy in the number of Na(+) molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na(+) binding sites.