Voltage and substrate dependence of the inverse transport mode of the rabbit Na⁺/glucose cotransporter (SGLT1)

Abstract

Properties of the cytoplasmic binding sites of the rabbit Na + /glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using K K-methyl D-glucopyranoside (K KMDG) as a substrate. The apparent affinity for K KMDG depends on the cytoplasmic Na + concentration and voltage. At 0 mV the K M for K KMDG is 7 mM at 110 mM Na + and 31 mM at 10 mM Na + . The apparent affinity for K KMDG and Na + is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half-maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.z 2000 Federation of European Biochemical Societies.

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

“…The present finding of a high apparent K i (> 1 mM) for phlorizin on the intracellular surface is in agreement with Sauer et al (2000). Phlorizin also inhibited Na + /sugar transport into inside-out membrane vesicles but only with a slow half-time (≈ 5 minutes).…”

“…For sugar activation, the obtained in the present study (32 ± 8 mM) is higher than 7.0 ± 0.8 mM obtained by Sauer et al (2000) under slightly different conditions (Na + -free external solutions). Our value is similar to that obtained for human SGLT1 (56 ± 9 mM) expressed in E. coli using inside-out membrane vesicle preparations with Na + -free external solutions .…”

“…Reverse sugar transport has also been studied for rabbit SGLT1 using the giant excised patch (Sauer et al, 2000). For sugar activation, the obtained in the present study (32 ± 8 mM) is higher than 7.0 ± 0.8 mM obtained by Sauer et al (2000) under slightly different conditions (Na + -free external solutions).…”

“…Computer simulations indicate that the 6-state model for SGLT1 can largely account for the steady-state kinetics of both forward and reverse modes. A preliminary account of this study has been presented (Eskandari, Loo & Wright, 1999), and since then a similar preliminary study has been reported (Sauer et al, 2000).…”

“…The currently available data ( Fig. 4; Sauer et al, 2000; may be fitted equally well with Hill coefficients of 1 or 2 and, therefore, the degree of co-operativity between the two internal binding sites remains to be resolved. Since the SGLT1 coupling of Na + to sugar transport stoichiometry is 2 (Parent et al 1992 a,b;Chen et al, 1995;Mackenzie et al, 1998), the most conservative interpretation of the results is that there is low cooperativity between the two Na + binding sites at the internal surface of the transporter.…”