Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Parent, Lucie; Supplisson, Stéphane; Loo, Donald D. F.; Wright, Ernest M.

doi:10.1007/bf00235797

Cited by 211 publications

(307 citation statements)

References 25 publications

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“…2D). Therefore, increasing the concentration of one substrate decreases the K m of the cosubstrate in G93T NIS, as reported for WT NIS (2) and other cotransporters (24,25).…”

Section: G93r Nis Issupporting

confidence: 75%

Mechanism of anion selectivity and stoichiometry of the Na ⁺ /I ^- symporter (NIS)

Paroder-Belenitsky¹,

Maestas

Dohán³

et al. 2011

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

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I − uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na þ ∕I − symporter (NIS) with an electrogenic 2Na þ : 1I − stoichiometry. We have obtained mechanistic information on NIS by characterizing the congenital I − transport defect-causing NIS mutant G93R. This mutant is targeted to the plasma membrane but is inactive. Substitutions at position 93 show that the longer the side chain of the neutral residue at this position, the higher the K m for the anion substrates. Unlike WT NIS, which mediates symport of Na þ and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 2∶1 stoichiometry. Furthermore, G93E/Q NIS discriminate between anion substrates, a discovery with potential clinical relevance. A 3D homology model of NIS based on the structure of the bacterial Na þ ∕galactose transporter identifies G93 as a critical player in the mechanism of the transporter: the changes from an outwardly to an inwardly open conformation during the transport cycle use G93 as a pivot.iodide transport defect | homology modeling | radioiodide therapy | sodium solute cotransporter family

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“…2D). Therefore, increasing the concentration of one substrate decreases the K m of the cosubstrate in G93T NIS, as reported for WT NIS (2) and other cotransporters (24,25).…”

Section: G93r Nis Issupporting

confidence: 75%

Mechanism of anion selectivity and stoichiometry of the Na ⁺ /I ^- symporter (NIS)

Paroder-Belenitsky¹,

Maestas

Dohán³

et al. 2011

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

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“…Phlorizin is a specific high-affinity competitive inhibitor of Na + /glucose cotransport from the external membrane surface, with an apparent inhibitory constant K i ≈ 10 µM and a true K i of 0.76 µM (Umbach, Coady & Wright, 1990;Birnir et al, 1991;Parent et al, 1992a;Hazama et al, 1997;Hirayama et al, 1996). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These protocols were approved by the UCLA Animal Research Committee, and meet all the NIH guidelines for the humane treatment of animals used for research. The oocytes were defolliculated, injected with 50 ng of rabbit SGLT1 cRNA, and maintained in Barth's medium (in mM: 88 NaCl, 1 KCl, 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 0.82 MgSO 4 , 2.4 NaHCO 3 , 10 HEPES, pH 7.4) supplemented with 5 µg/ml gentamycin at 18°C for 3-8 days until use (Birnir, Loo & Wright, 1991;Parent et al, 1992a;Loo et al, 1993).…”

Section: Oocyte Preparationmentioning

confidence: 99%

“…For transport in the forward direction, we used the two-electrode voltage clamp on intact oocytes and the sugar-dependent currents were measured as previously described (Birnir et al, 1991;Parent et al, 1992a;Loo et al, 1993). The oocytes were normally bathed in a NaCl buffer (in mM: 100 NaCl, 2 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 HEPES, pH 7.5).…”

Section: Sugar Transportmentioning

confidence: 99%

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

2005

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This study investigates the reverse mode of the Na + /glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na + -and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The currentvoltage relationship saturated at positive membrane voltages (30-50 mV), and approached zero at − 150 mV. The half-maximal concentration for αMDG-evoked outward current ( ) was 35 mM (at 0 mV). In comparison, for forward sugar transport was 0.15 mM (at 0 mV). was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-Omethyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na + /sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.

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“…Maximal charge (Q max ) was conserved upon progressive removal of Na ϩ , whereas the voltage midpoint (V 0.5 ) shifted to more hyperpolarized potentials. Pre-steady-state currents have been observed for several other Na ϩ -coupled (1,9,14,17,19,27) or H ϩ -coupled (8,12,16,21,22,25) solute transporters. The results of model simulation have led investigators to attribute pre-steady-state currents to two steps in the transport cycle, namely reorientation of the empty charged carrier and binding/dissociation of the driving ion partway within the plane of the membrane electric field (and in some cases also to the binding/dissociation of the ion at the internal face).…”

Section: Discussionmentioning

confidence: 99%

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Mackenzie¹,

Illing²,

Hediger³

2008

American Journal of Physiology-Cell Physiology

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Mackenzie B, Illing AC, Hediger MA. Transport model of the human Na ϩ -coupled L-ascorbic acid (vitamin C) transporter SVCT1. Am J Physiol Cell Physiol 294: C451-C459, 2008. First published December 19, 2007 doi:10.1152/ajpcell.00439.2007.-Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-Ascorbic acid transport was saturable (K0.5 Ϸ 70 M), temperature dependent (Q10 Ϸ 5), and energized by the Na ϩ electrochemical potential gradient. We obtained a Na ϩ -L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-Ascorbic acid and Na ϩ saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na ϩ , L-ascorbic acid, Na ϩ . In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na ϩ , maximal charge translocation (Qmax) was Ϸ25 nC, around a midpoint (V0.5) at Ϫ9 mV, and with apparent valence ϷϪ1. Qmax was conserved upon progressive removal of Na ϩ , whereas V0.5 shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na ϩ partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease. cotransporters; sodium dependent; intestinal absorption; model simulation; renal reabsorption; Xenopus oocyte VITAMIN C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant scavenger of free radicals and as a cofactor in many enzymatic reactions (3,6,10,26,29). It cannot be synthesized in Homo sapiens and must be derived from the diet. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial Na ϩ -dependent Lascorbic acid cotransporter SVCT1 (reviewed in Refs. 32 and 35), the product of the SLC23A1 gene. Selective sorting of SVCT1 protein to the apical membrane has been demonstrated in cultured cell lines (Caco2 and MDCK) of intestinal and renal origin (2, 23).We and others previously demonstrated that human SVCT1 is a Na ϩ -dependent transporter that favors L-ascorbic acid over D-isoascorbic acid and that the oxidized form of vitamin C, dehydroascorbic acid, is excluded (5, 34). Here we explored its molecular mechanisms by testing the hypothesis that the Na ϩ dependence and rheogenicity of SVCT1 arise from...

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Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Cited by 211 publications

References 25 publications

Mechanism of anion selectivity and stoichiometry of the Na ⁺ /I ^- symporter (NIS)

Mechanism of anion selectivity and stoichiometry of the Na ⁺ /I ^- symporter (NIS)

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

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1

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Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Cited by 211 publications

References 25 publications

Mechanism of anion selectivity and stoichiometry of the Na + /I - symporter (NIS)

Mechanism of anion selectivity and stoichiometry of the Na + /I - symporter (NIS)

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

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

Contact Info

Product

Resources

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Mechanism of anion selectivity and stoichiometry of the Na ⁺ /I ^- symporter (NIS)

Mechanism of anion selectivity and stoichiometry of the Na ⁺ /I ^- symporter (NIS)

Transport model of the human Na⁺-coupled l-ascorbic acid (vitamin C) transporter SVCT1