Kinetics of Steady-state Currents and Charge Movements Associated with the Rat Na+/Glucose Cotransporter

Abstract

The rat Na ؉ /glucose cotransporter (SGLT1) was expressed in Xenopus oocytes and steady-state and transient currents were measured using a two-electrode voltage clamp. The maximal glucose induced Na ؉ -dependent inward current was ϳ300 -500 nA. The apparent affinity constants for sugar (␣-methyl-D-glucopyranoside; ␣MDG) (K 0.5 ␣MDG ) and sodium (K 0.5 Na ) at a membrane potential of ؊150 mV were 0.2 mM and 4 mM. The K 0.5 Na increased continuously with depolarizing potentials reaching 40 mM at ؊30 mV. K 0.5 ␣MDG was steeply voltage dependent, 0.46 mM at ؊30 mV and 1 mM at ؊10 mV. From all tested monovalent cations only Li ؉ could substitute for Na ؉ , but with lower affinity. The relative substrate specificity was D-glucose > ␣MDG Ϸ D-galactose > 3-OMe-Glc > > ␤-naphthyl-D-glucoside > > uridine. Phlorizin (Pz), the specific blocker of sugar transport, showed an extremely high affinity for the rat cotransporter with an inhibitor constant (K i Pz ) of 12 nM. SGLT1 charge movements in the absence of sugar were fitted by the Boltzmann equation with an apparent valence of the movable charge of ϳ1, a potential for 50% maximal charge transfer (V 0.5 ) of ؊43 mV, and a maximal charge (Q max ) of 9 nanocoulombs. The apparent turnover number for the rat SGLT1 was 30 s ؊1. Model simulations showed that the kinetics of the rat SGLT1 are described by a six-state ordered nonrapid equilibrium model, and comparison of the kinetics of the rat, rabbit and human cotransporters indicate that they differ mainly in their presteady-state kinetic parameters.Cotransporters are membrane proteins that use the electrochemical potential gradient for ions to accumulate sugars, amino acids and osmolytes into cells. Using the electrochemical potential gradient for Na ϩ , the Na ϩ /glucose cotransporter (SGLT) 1 accumulates glucose across the brush border membrane of the epithelial cells of the intestine and the proximal tubule of the kidney.Several members of the SGLT family have been cloned, and these include the high affinity glucose cotransporters (SGLT1, K 0.5 ␣MDG ϳ 0.2 mM) from rabbit small intestine (1) and kidney (2), pig (3), and rat kidney (4), human intestine (5), as well as the low affinity glucose cotransporter (pSGLT2, K 0.5 ␣MDG ϳ2 mM) from pig kidney (6). Mapping the genomic arrangement of the human SGLT1 gene, Turk et al. (7) showed that SGLT1 is a single-copy gene, so that the amino acid sequences from various tissues of a given species are identical. Comparison of the amino acid sequences from the rat, human and rabbit clones reveal 86 -87% identity and 93-94% similarity. How does this high degree of homology between the three clones affect their kinetic properties? In this study, we characterized the presteady-state and steady-state kinetics of the rat SGLT1 clone with a view to understand the relationship between structure and function of members of the SGLT1 family. MATERIALS AND METHODSThe pBluescript II SK plasmid containing the coding sequence for rat SGLT1 (4) was linearized with SalI and transcribed in vitro with T3 ...

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

“…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).…”

An Emerging Protagonist: Sodium Glucose Co-transporters (SGLTs) as a Burgeoning Target for the Treatment of Diabetes Mellitus

“…But there are several limitation of this SGLT inhibitor is that it is non-selective SGLT inhibitor, inhibiting both SGLT2 and SGLT1 inhibitor [14,15].…”

Arginine‐427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane

“…The plasmid carrying the cDNA sequence for rbSGLT1 [1] was used as template to perform oligonucleotide-directed mutagenesis [2]. The sequence of the synthetic oligonucleotide used to introduce the R427A mutation in its 5'~3' direction was: CAGGAACAGCATGAACAAC-GCTCC TGCGATCATGA (mutated nucleotides in bold letters).…”

“…For example, mutation of aspartic acid-176 to alanine (D176A) in tr~msmembrane domain M4 of the rabbit Na+/glucose cotranspc, rter (rbSGLT1) alters the kinetics of charge transfer without changing the steady-state kinetics [1]. The interpretation of su=h experiments can be more complex because mutations can aher translation, processing and trafficking from the endoplasm~c reticulum to the plasma membrane.…”