Kinetic analysis of mechanism of intestinal Na+-dependent sugar transport

Restrepo, Diego; Kimmich, George A.

doi:10.1152/ajpcell.1985.248.5.c498

Cited by 37 publications

(43 citation statements)

References 15 publications

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“…The 1-min ['4C] aMeGlc uptake was within the linear range ofuptake and was 19±0.8 pmol/oocyte per min (n = 8). The Na+ to glucose coupling ratio for SGLT1 is therefore 2:1, consistent with the studies of SGLT1 expressed in oocytes ( 14) and with the known stoichiometry of the high affinity Na+/ glucose cotransport in brush border membrane vesicles ( 12,30,31).…”

Section: Resultssupporting

confidence: 86%

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

Kanai¹,

et al. 1994

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The major reabsorptive mechanism for D-glucose in the kidney is known to involve a low affinity high capacity Na+/glucose cotransporter, which is located in the early proximal convoluted tubule segment S1, and which has a Na' to glucose coupling ratio of 1:1. Here we provide the first molecular evidence for this renal D-glucose reabsorptive mechanism. We report the characterization of a previously cloned human kidney cDNA that codes for a protein with 59% identity to the high affinity Na+/glucose cotransporter (SGLT1). Using expression studies with Xenopus laevis oocytes we demonstrate that this protein (termed SGLT2) mediates saturable Na'-dependent and phlorizin-sensitive transport of D-glucose and a-methyl-D-glucopyranoside (aMeGIc) with K. values of 1.6 mM for aMeGlc and -250 to 300 mM for Na', consistent with low affinity Na+/glucose cotransport. In contrast to SGLT1, SGLT2 does not transport D-galactose. By comparing the initial rate of I 4Cl-aMeGIc uptake with the Na'-influx calculated from aMeGIcevoked inward currents, we show that the Na+ to glucose coupling ratio of SGLT2 is 1:1. Using combined in situ hybridization and immunocytochemistry with tubule segment specific marker antibodies, we demonstrate an extremely high level of SGLT2 message in proximal tubule S1 segments. This level of expression was also evident on Northern blots and likely confers the high capacity of this glucose transport system. We conclude that SGLT2 has properties characteristic of the renal low affinity high capacity Na+/glucose cotransporter as previously reported for perfused tubule preparations and brush border membrane vesicles. Knowledge of the structural and functional properties of this major renal Na+/glucose reabsorptive mechanism will advance our understanding of the pathophysiology of renal diseases such as familial renal glycosuria and diabetic renal disorders. (J. Clin. Invest. 1994. 93:397-404.)

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Section: Resultssupporting

confidence: 86%

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

Kanai¹,

et al. 1994

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“…Most investigations of sodium-coupled cotransport systems have been carried out so far by measuring isotope fluxes in membrane vesicles or intact cells (Kessler & Semenza, 1983;Turner, 1983;Koepsell et al, 1984;Restrepo & Kimmich, 1985). In this study we have used the whole-cell recording technique for measuring electric currents associated with sodium-driven alanine transport.…”

Section: Discussionmentioning

confidence: 99%

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings

1986

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Electrical currents associated with sodium-coupled alanine transport in mouse pancreatic acinar cells were studied using the method of whole-cell recording with patch pipettes. Single cells or small clusters of (electrically coupled) cells were isolated by collagenase treatment. The composition of the intracellular solution could be controlled by internal perfusion of the patch pipette. In this way both inward and outward currents could be measured under "zero-trans" conditions, i.e., with finite concentrations of sodium and L-alanine on one side and zero concentrations on the other. Inward and outward currents for equal but opposite concentration gradients were found to be of similar magnitude, meaning that the cotransporter is functionally nearly symmetric. The dependence of current on the concentrations of sodium and L-alanine exhibited a Michaelis-Menten behavior. From the sodium-concentration dependence of current as well as from the reversal potential of the current in the presence of an alanine-concentration gradient, a sodium/alanine stoichiometric ratio of 1:1 can be inferred. The finding that N-methylated amino acids may substitute for L-alanine, as well as the observed pH dependence of currents indicate that the pancreatic alanine transport system is similar to (or identical with) the "A-system" which is widespread in animal cells. The transport system is tightly coupled with respect to Na+; alanine-coupled inward flow of Na+ is at least 30 times higher than uncoupled Na+ flow mediated by the cotransporter. The current-voltage characteristic of the cotransporter could be (approximately) determined from the difference of transmembrane current in the presence and in the absence of L-alanine. The sodium-concentration dependence of the current-voltage characteristic indicates that a Na+ ion approaching the binding site from the extracellular medium has to cross part of the transmembrane electric field.

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“…At least two different classes of mechanisms of ioncoupled cotransport are feasible (Schultz & Curran, 1970;Stein, 1976;Crane & Dorando, 1980;Eddy, 1980;Hopfer & Groseclose, 1980;Turner & Silverman, 1980;Turner, 1981Turner, , 1983Harrison, Rove, Lumsden & Silverman, 1984;Sanders, Hansen, Gradmann & Slayman, 1984;Semenza, Kessler, Hosang, Weber & Schmidt, 1984;Restrepo & Kimmich, 1985a;Sanders, 1986;Wright, 1986). In the "simultaneous" mechanism the ion N and the substrate S bind to the transporter C, forming a ternary complex NCS which alternates between states NC'S and NC"S with inward-facing and outwardfacing binding sites.…”

Section: Microscopic Models For Ion-driven Substrate Transportmentioning

confidence: 99%

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Jauch

Läuger

1986

J. Membrain Biol.

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In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the "simultaneous" mechanism the cotransporter C forms a ternary complex NCS with Na+ and the substrate S; coupled transport of Na+ and S involves a conformational transition between states NC'S and NC"S with inward- and outward-facing binding sites. In the "consecutive" (or "ping-pong") mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the "simultaneous" model, but incompatible with the "consecutive" mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the "simultaneous" model can be estimated from the experimental results. The equilibrium dissociation constants of Na+ and alanine at the extracellular side are determined to be K"N less than or equal to 64 mM and K"S less than or equal to 18 mM. Furthermore, the ratio K"N/KS"N of the dissociation constants of Na+ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be less than or equal to 6. This indicates that the binding sequence of Na+ and S to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na+ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.

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Kinetic analysis of mechanism of intestinal Na+-dependent sugar transport

Cited by 37 publications

References 15 publications

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

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