Sugar Transport Heterogeneity in the Kidney: Two Independent Transporters or Different Transport Modes through an Oligomeric Protein? 1. Glucose Transport Studies

Abstract

The kinetics of Na+/d-glucose cotransport (SGLT) were reevaluated in rabbit renal brush border membrane vesicles isolated from the whole kidney cortex using a fast-sampling, rapid-filtration apparatus (FSRFA, US patent #5,330,717) for uptake measurements. Our results confirm SGLT heterogeneity in this preparation, and both high (HAG) and low (LAG) affinity glucose transport pathways can be separated over the 15-30 degrees C range of temperatures. It is further shown that: (i) Na+ is an essential activator of both HAG and LAG; (ii) similar energies of activation can be estimated from the linear Arrhenius plots constructed from the Vmax data of HAG and LAG, thus suggesting that the lipid composition and/or the physical state of the membrane do not affect much the functioning of SGLT; (iii) similar Vmax values are observed for glucose and galactose transport through HAG and LAG, thus demonstrating that the two substrates share the same carrier agencies; and (iv) phlorizin inhibits both HAG and LAG competitively and with equal potency (Ki = 15 microM). Individually, these data do not allow us to resolve conclusively whether the kinetic heterogeneity of SGLT results from the expression in the proximal tubule of either two independent transporters (rSGLT1 and rSGLT2) or from a unique transporter (rSGLT1) showing allosteric kinetics. Altogether and compared to the kinetic characteristics of the cloned SGLT1 and SGLT2 systems, they do point to a number of inconsistencies that lead us to conclude the latter possibility, although it is recognized that the two alternatives are not mutually exclusive. It is further suggested, from the differences in the Km values of HAG transport in the kidney as compared to the small intestine and SGLT1 cRNA-injected oocytes, that renal SGLT1 activity is somehow modulated, maybe through heteroassociation with (a) regulatory subunit(s) that might also contribute quite significantly to sugar transport heterogeneity in the kidney proximal tubule.

Pharmacokinetic and Pharmacodynamic Modeling of the Effect of an Sodium-Glucose Cotransporter Inhibitor, Phlorizin, on Renal Glucose Transport in Rats

“…diger and Rhoads, 1994;Wright, 2001) and different properties in affinity and capacity for glucose transport between SGLT1 and SGLT2 (Turner and Moran, 1982;Oulianova and Berteloot, 1996). The present model suitably expressed the relationship between plasma glucose level and renal glucose clearance (Fig.…”

“…Two isoforms of SGLT with different affinity for glucose are localized in different positions in the proximal tubule; a low-affinity transporter (SGLT2) is expressed in the convoluted tubule, and a high-affinity transporter (SGLT1) is expressed in the straight tubule (Hediger and Rhoads, 1994;Wright, 2001). In vitro studies using renal brush-border membrane vesicles (BBMV) (Turner and Moran, 1982;Oulianova and Berteloot, 1996) indicate that the low-affinity transporter has high capacity for glucose transport and that the high-affinity one has low capacity.…”

Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells

“…Phlorizin, an aromatic ␤-glucoside, is a well-known potent competitive inhibitor of the SGLT1 transporter (Diedrich, 1966). It is postulated that phlorizin is supposed to bind with a two-step mechanism to both the sugar-binding site of the transporter (mainly through hydrogen bonds) and to a binding site for the aglucone moiety (phloretin) mainly through hydrophobic interactions Oulianova and Berteloot, 1996;Oulianova et al, 2001). Site-directed mutagenesis studies and studies using reconstituted peptide in vitro have shown that the C-terminal loop 13 is involved in the binding of phlorizin (Novakova et al, 2001;Raja et al, 2003;Xia et al, 2003).…”

Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling

“…It is proposed that the phlorizin binding to SGLT1 is a two-step process: rapid formation of an initial collision complex, followed by a slow isomerization process that occludes phlorizin within its receptor site (8). Phlorizin is thereby supposed to bind to both the sugar-binding site and the aglucon-binding site, the latter with a hydrophobic/aromatic surface (9,10).…”