Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

Eskandari, Sepehr; Wright, Ernest M.; Loo, Donald D. F.

doi:10.1007/s00232-005-0743-x

Cited by 64 publications

(100 citation statements)

References 27 publications

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“…Although we hesitate to make solid estimates of the Na + K d value to this state, these counts suggest a value greater than 150 mM, which is even weaker than the K M of 12 mM estimated for hSGLT1 (26). Last, in our previous simulations, we computed the galactose binding free energy to the inward-facing state to be 1-2 k B T (7), again consistent with the weak K d value reported for rabbit SGLT1 (27,28).…”

Section: Resultssupporting

confidence: 72%

Stochastic steps in secondary active sugar transport

Adelman

Ghezzi

Bisignano

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Secondary active transporters, such as those that adopt the leucinetransporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.T ransport of sugar molecules across membranes is virtually ubiquitous in biology, and it is of central importance in human health. The uptake of glucose is especially crucial due to its pivotal role in cellular metabolism and energy production. In mammals, glucose is absorbed in the small intestine and kidney via sodium-dependent glucose transporters (SGLTs), which localize to the apical membrane and concentrate glucose in the epithelia. SGLTs fall into the large leucine-transporter (LeuT) structural family of secondary active transporters that have evolved to concentrate a wide array of substrates across membranes using the energy stored in the Na + electrochemical potential gradient. For symporters in this family, transport occurs by an alternating access mechanism (1) in which the transporter first binds ligands in an outward-facing conformation, and then transitions to an inwardfacing conformation that releases the cargo to the cytoplasm. The order of ion and substrate binding and unbinding is likely tied to the function of the transporter, making it possible to convert the energy stored in the ion gradient into a substrate gradient, and vice versa when these proteins operate in reverse.Extensive biochemical uptake assays and electrophysiological studies of SGLTs have led to a view of Na + /glucose cotransport in which Na + binding precedes sugar binding on the external face of the transporter. Kinetic models adhering to this mechanism satisfactorily account fo...

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Section: Resultssupporting

confidence: 72%

Stochastic steps in secondary active sugar transport

Adelman

Ghezzi

Bisignano

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…A canonical nonselective SGLT inhibitor, phlorizin, the parent compound of selective SGLT2 inhibitors, exhibits a ∼600-fold lower inhibition constant (K i ) value on SGLT1 from the extracellular side than it does from the intracellular side (Eskandari et al, 2005). Phlorizin is freely filtrated by the glomerulus and mainly excreted into urine, which is in line with the idea that the glomerular-filtrated phlorizin acts on SGLTs from the luminal side to increase urinary glucose excretion (Silverman, 1974).…”

Section: Introductionsupporting

confidence: 62%

Interaction of the Sodium/Glucose Cotransporter (SGLT) 2 inhibitor Canagliflozin with SGLT1 and SGLT2

Ohgaki

Wei

Yamada

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Canagliflozin, a selective sodium/glucose cotransporter (SGLT) 2 inhibitor, suppresses the renal reabsorption of glucose and decreases blood glucose level in patients with type 2 diabetes. A characteristic of canagliflozin is its modest SGLT1 inhibitory action in the intestine at clinical dosage. To reveal its mechanism of action, we investigated the interaction of canagliflozin with SGLT1 and SGLT2. Inhibition kinetics and transporter-mediated uptake were examined in human SGLT1-or SGLT2-expressing cells. Whole-cell patch-clamp recording was conducted to examine the sidedness of drug action. Canagliflozin competitively inhibited SGLT1 and SGLT2, with high potency and selectivity for SGLT2. Inhibition constant (K i ) values for SGLT1 and SGLT2 were 770.5 and 4.0 nM, respectively. 14 C-canagliflozin was suggested to be transported by SGLT2; however, the transport rate was less than that of a-methyl-D-glucopyranoside. Canagliflozin inhibited a-methyl-D-glucopyranoside-induced SGLT1-and SGLT2-mediated inward currents preferentially from the extracellular side and not from the intracellular side. Based on the K i value, canagliflozin is estimated to sufficiently inhibit SGLT2 from the urinary side in renal proximal tubules. The K i value for SGLT1 suggests that canagliflozin suppresses SGLT1 in the small intestine from the luminal side, whereas it does not affect SGLT1 in the heart and skeletal muscle, considering the maximal concentration of plasma-unbound canagliflozin. Similarly, SGLT1 in the kidney would not be inhibited, thereby aiding in the prevention of hypoglycemia. After binding to SGLT2, canagliflozin may be reabsorbed by SGLT2, which leads to the low urinary excretion and prolonged drug action of canagliflozin.

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“…Experiments done on individual partial reactions in the model have refined these estimates (14,29,47). We have used the most recent set of rate constants reviewed in Ref.…”

Section: Sglt1mentioning

confidence: 99%

Transepithelial glucose transport and Na⁺/K⁺ homeostasis in enterocytes: an integrative model

Thorsen

Drengstig

Ruoff

2014

American Journal of Physiology-Cell Physiology

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Thorsen K, Drengstig T, Ruoff P. Transepithelial glucose transport and Na ϩ /K ϩ homeostasis in enterocytes: an integrative model. Am J Physiol Cell Physiol 307: C320 -C337, 2014. First published June 4, 2014; doi:10.1152/ajpcell.00068.2013.-The uptake of glucose and the nutrient coupled transcellular sodium traffic across epithelial cells in the small intestine has been an ongoing topic in physiological research for over half a century. Driving the uptake of nutrients like glucose, enterocytes must have regulatory mechanisms that respond to the considerable changes in the inflow of sodium during absorption. The Na-K-ATPase membrane protein plays a major role in this regulation. We propose the hypothesis that the amount of active Na-K-ATPase in enterocytes is directly regulated by the concentration of intracellular Na ϩ and that this regulation together with a regulation of basolateral K permeability by intracellular ATP gives the enterocyte the ability to maintain ionic Na ϩ /K ϩ homeostasis. To explore these regulatory mechanisms, we present a mathematical model of the sodium coupled uptake of glucose in epithelial enterocytes. Our model integrates knowledge about individual transporter proteins including apical SGLT1, basolateral Na-K-ATPase, and GLUT2, together with diffusion and membrane potentials. The intracellular concentrations of glucose, sodium, potassium, and chloride are modeled by nonlinear differential equations, and molecular flows are calculated based on experimental kinetic data from the literature, including substrate saturation, product inhibition, and modulation by membrane potential. Simulation results of the model without the addition of regulatory mechanisms fit well with published short-term observations, including cell depolarization and increased concentration of intracellular glucose and sodium during increased concentration of luminal glucose/sodium. Adding regulatory mechanisms for regulation of Na-K-ATPase and K permeability to the model show that our hypothesis predicts observed long-term ionic homeostasis.enterocytes; epithelial transport; homeostasis; mathematical modeling; ionic regulation THE UPTAKE AND TRANSPORT OF glucose by the epithelial enterocytes in the small intestine are essential steps in all higher animals. Much of the glucose uptake in the small intestine is coupled to transport of sodium, and the different transporter proteins involved in the sodium coupled glucose uptake are well known (23,61,67,97). Although the electrochemical kinetics of the individual transporter proteins responsible for glucose and nutrient uptake are fairly elucidated (19,54,67,82,97), the combined function in which these transporters drive a net inflow of nutrients from the intestinal lumen to the serosal blood has not yet been modeled in detail.We have developed a mathematical model of an enterocyte that incorporates the available kinetic data from studies on individual transporter proteins. The model includes apical SGLT1 (the glucose sodium cotransporter), coupled apical flow of Na ϩ an...

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Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

Cited by 64 publications

References 27 publications

Stochastic steps in secondary active sugar transport

Stochastic steps in secondary active sugar transport

Interaction of the Sodium/Glucose Cotransporter (SGLT) 2 inhibitor Canagliflozin with SGLT1 and SGLT2

Transepithelial glucose transport and Na⁺/K⁺ homeostasis in enterocytes: an integrative model

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Kinetics of the Reverse Mode of the Na+/Glucose Cotransporter

Cited by 64 publications

References 27 publications

Stochastic steps in secondary active sugar transport

Stochastic steps in secondary active sugar transport

Interaction of the Sodium/Glucose Cotransporter (SGLT) 2 inhibitor Canagliflozin with SGLT1 and SGLT2

Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model

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Transepithelial glucose transport and Na⁺/K⁺ homeostasis in enterocytes: an integrative model