Kinetic and specificity differences between rat, human, and rabbit Na+-glucose cotransporters (SGLT-1)

Abstract

The Na+ activation and substrate specificity of human, rabbit, and rat Na+-glucose cotransporter (SGLT-1) isoforms were characterized using the Xenopus oocyte expression system and the two-electrode voltageclamp method. We find that there are differences, major and minor, in both the kinetics and substrate specificities between these isoforms; the substrate concentration at half-maximal current (K0.5) for hexoses varies from 0.2 to > 40 mM, depending on the species and sugar; the affinity constant (Ki) for phlorizin, the classic competitive inhibitor of SGLT-1, varies lover two orders of magnitude (rat Ki = 0.03 microM vs. rabbit Ki = 1.4 microM); and some glucoside inhibitors of the rabbit isoform, p-nitrophenyl glucose and beta-naphthyl glucose, are transported by the human and rat transporters. Na+ activation is more sensitive to membrane potential in the human and rat isoforms compared with rabbit. The rabbit isoform has a higher apparent affinity for alpha-methylglucose and 3-O-methylglucose by a factor of two than either human or rat. These results can be quantitatively fitted by our six-state kinetic model of SGLT-1, providing insight into the processes involved in these changes. For example, the model predicts that Na+ binding (rate constant, k12) in human and rat SGLT-1 is similar but is fourfold larger than in rabbit, whereas sugar binding (k23) in rabbit and rat is similar but double the value in human SGLT-1. The differences in the primary amino acid sequences between these three homologous proteins must account for the kinetic and substrate specificity differences, and comparisons of the functional properties and amino acid sequences of SGLT-1 isoforms provide useful information about structure/function relationships.

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

“…Similar results were obtained for sugar competition of αMDG uptake into inside-out membrane vesicles expressing hSGLT1 (αMDG > 3-O-methyl-glucose ~ D-galactose > D-glucose; Quick & Tomasevic & Wright, 2003). In contrast, the relative affinities for sugar cotransport in the forward direction by rabbit, rat and human isoforms of SGLT1 is αMDG ~ D-glucosẽ D-galactose > 3-O-methyl-glucose (Ikeda et al, 1989;Birnir et al, 1991;PanayatovaHeiermann, Loo & Wright, 1995;Hirayama et al, 1996;Díez-Sampedro et al, 2001). The differences in sugar affinity and selectivity for cotranport in the two directions under the same driving forces suggest differences in the architecture of the outward-and inwardfacing sugar binding sites.…”

“…The apparent affinity constant for sugar is 250-fold higher for the reverse mode than that for the forward mode (at 0 mV, is 0.15 mM for the forward, and 35 mM for the reverse mode). The asymmetry in sugar affinity is accompanied by a similar loss of affinity for phlorizin between both membrane surfaces: the true K i for phlorizin increased from 0.76 µM on the external surface (Hirayama et al, 1996) to ≈ 0.5 mM on the internal surface. Sugar specificity also differs between the forward and reverse modes.…”

“…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.…”

“…Membrane voltage influences the conformational change of the empty transporter (between C 1 and C 6 ), and Na + binding/dissociation (between C 1 and C 2 ). Computer simulations of forward sugar transport have yielded sets of rate constants that account for the presteady-state and steadystate kinetics of rabbit and human SGLT1 (Parent et al, 1992b;Loo et al, 1993Loo et al, ,2002Panayotova-Heiermann, 1995;Hirayama et al, 1996;Hazama et al, 1997).…”

The Plasma Membrane-associated Protein RS1 Decreases Transcription of the Transporter SGLT1 in Confluent LLC-PK1 Cells

“…7, V max ϭ 2.03 Ϯ 0.05 nmol ϫ mg decreasing membrane potential (46), the observed difference in K m may be due to the stronger depolarization in the more active native LLC-PK 1 cells. The apparent K m value for AMG uptake by human SGLT1 expressed in Xenopus oocytes was increased by a similar factor when the membrane potential was decreased from Ϫ70 to Ϫ30 mV (46). To determine whether the repression of SGLT1 expression after cultivation with high D-glucose was preserved after overexpression of pRS1, cell lines S11 and S12 were grown with 5 or 25 mM D-glucose and harvested 5 days after confluence, and phlorizin-inhibitable uptake of 10 M AMG was measured after 2.5 min of incubation.…”