Biophysical Characteristics of the Pig Kidney Na+/Glucose Cotransporter SGLT2 Reveal a Common Mechanism for SGLT1 and SGLT2

Abstract

The Na ؉ -dependent, low affinity glucose transporter SGLT2 cloned from pig kidney is 76% identical (at the amino acid level) to its high affinity homologue SGLT1. Using two-microelectrode voltage clamp, we have characterized the presteady-state and steady-state kinetics of SGLT2 expressed in Xenopus oocytes. The kinetic properties of the steady-state sugar-evoked currents as a function of external Na ؉ and ␣-methyl-D-glucopyranoside (␣MG) concentrations were consistent with an ordered, simultaneous transport model in which Na ؉ binds first. Na ؉ binding was voltage-dependent and saturated with hyperpolarizing voltages. Phlorizin was a potent inhibitor of the sugar-evoked currents (K i Pz Ϸ 10 M) and blocked an inward Na ؉ current in the absence of sugar. SGLT2 exhibited Na ؉ -dependent presteadystate currents with time constants 3-7 ms. Charge movements were described by Boltzmann relations with apparent valence Ϸ 1 and maximal charge transfer Ϸ 11 nC, and were reduced by the addition of sugar or phlorizin. The differences between SGLT1 and SGLT2 were that (i) the apparent affinity constant (K 0.5 ) for ␣MG (Ϸ3 mM) was an order of magnitude higher for SGLT2; (ii) SGLT2 excluded galactose, suggesting discrete sugar binding; (iii) K 0.5 for Na ؉ was lower in SGLT2; and (iv) the Hill coefficient for Na ؉ was 1 for SGLT2 but 2 for SGLT1. Simulations of the six-state kinetic model previously proposed for SGLT1 indicated that many of the kinetic properties observed in SGLT2 are expected by simply reducing the Na ؉ /glucose coupling from 2 to 1.In the proximal tubule of the kidney, reabsorption of filtered glucose at the apical membrane is mediated by one or more Na ϩ /glucose cotransporters. The low affinity glucose transporter (SGLT2) 1 (1), cloned from a pig kidney cell line (2), bears 76% identity at the amino acid level to its high affinity homologue SGLT1 (3), yet exhibits functional characteristics (1) that clearly distinguish it from SGLT1.In the present study we examined the mechanisms of Na ϩ / glucose cotransport via the pig SGLT2 transporter expressed in Xenopus oocytes. We determined the dependence of the steadystate kinetics on Na ϩ and ␣MG concentrations, and examined presteady-state charge movements associated with SGLT2. We have also extended our analysis of the substrate selectivity of SGLT2, its cation activation, and the effects of phlorizin upon both the uncoupled and the sugar-coupled Na ϩ pathways through the transporter. EXPERIMENTAL PROCEDURESStage V-VI oocytes from Xenopus laevis were injected with pSGLT2 cRNA as described previously (1). Electrophysiological experiments were performed using a two-microelectrode voltage clamp (1, 4, 5). Oocytes were superfused at 20 -22°C with experimental medium in which the Na ϩ concentration varied between 0 and 100 mM by equimolar replacement with choline or Li ϩ . Test solutions (additionally containing sugars and/or phlorizin) were always washed out by superfusing the oocyte with substrate-free, choline chloride medium (pH 7.5). Sugar-evoked currents (I...

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

“…3D), indicating that the second Na ϩ must bind after the ascorbate-binding step. In contrast, considering a carrier model in dynamic equilibrium, we should expect that saturating L-ascorbic acid would always drive the transporter at its maximal rate were L-ascorbic acid bound last (11,17,19,31). Our data are not consistent with a random-binding model in which case I max for either substrate would be independent of the cosubstrate concentration.…”

“…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).…”

“…B: comparison of Q Asc (gray bar) and 22 Na ϩ accumulation (filled bar) over 5 min in oocytes expressing SVCT1 (mean Ϯ SD, n ϭ 6) superfused with 50 mM 22 Na ϩ plus 1 mM L-ascorbic acid, after subtraction of Q Asc (19.3 Ϯ 5.7 pmol) and 22 Na ϩ accumulation (157 Ϯ 294 pmol) measured in control oocytes (n ϭ 3). The Na ϩ /Q Asc ratio was 2.0 Ϯ 0. higher concentrations of the cosubstrate (11,17,19,31). In contrast, for a consecutive carrier model, the K 0.5 values are expected to rise as cosubstrate concentration is increased.…”

“…We did not consider the inward-facing Na ϩ -bound configuration C7 ϭ [CNa]Љ since intracellular Na ϩ concentration ([Na ϩ ]) is generally low. Our partial-reaction model is essentially identical to that used to describe the Na ϩ -glucose cotransporters SGLT1 (9,14,28) and SGLT3 (formerly pSGLT2) (17) and similar to that used to describe a myo-[H ϩ ]inositol cotransporter (12). The rate of change in concentration of each carrier state is given by the difference of forward and reverse reactions…”

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa

“…6c). As there is a close structural relationship between the proteins of the SGLT family (Mackenzie et al, 1996), recognition of all of these proteins by the same polyclonal antibody is likely, thereby indicating that the sperm proteins which recognize the SGLT-1 antibody are probably members of the SGLT family of transporters.…”