A Single Amino Acid Change Converts the Sugar Sensor SGLT3 into a Sugar Transporter

Background

Sodium-glucose cotransporter proteins (SGLT) belong to the SLC5A family, characterized by the cotransport of Na⁺ with solute. SGLT1 is responsible for intestinal glucose absorption. Until recently the only role described for SGLT proteins was to transport sugar with Na⁺. However, human SGLT3 (hSGLT3) does not transport sugar but causes depolarization of the plasma membrane when expressed in Xenopus oocytes. For this reason SGLT3 was suggested to be a sugar sensor rather than a transporter. Despite 70% amino acid identity between hSGLT3 and hSGLT1, their sugar transport, apparent sugar affinities, and sugar specificity differ greatly. Residue 457 is important for the function of SGLT1 and mutation at this position in hSGLT1 causes glucose-galactose malabsorption. Moreover, the crystal structure of vibrio SGLT reveals that the residue corresponding to 457 interacts directly with the sugar molecule. We thus wondered if this residue could account for some of the functional differences between SGLT1 and SGLT3.

Methodology/Principal Findings

We mutated the glutamate at position 457 in hSGLT3 to glutamine, the amino acid present in all SGLT1 proteins, and characterized the mutant. Surprisingly, we found that E457Q-hSGLT3 transported sugar, had the same stoichiometry as SGLT1, and that the sugar specificity and apparent affinities for most sugars were similar to hSGLT1. We also show that SGLT3 functions as a sugar sensor in a living organism. We expressed hSGLT3 and E457Q-hSGLT3 in C. elegans sensory neurons and found that animals sensed glucose in an hSGLT3-dependent manner.

Conclusions/Significance

In summary, we demonstrate that hSGLT3 functions as a sugar sensor in vivo and that mutating a single amino acid converts this sugar sensor into a sugar transporter similar to SGLT1.     

Missense mutations in SGLT1 cause glucose–galactose malabsorption by trafficking defects

Glucose–galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na⁺/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [¹⁴C]α-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.     

Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.     

Protein kinase‐A affects sorting and conformation of the sodium‐dependent glucose co‐transporter SGLT1

In Chinese hamster ovary cells expressing rabbit sodium‐dependent glucose transporter (rbSGLT1) protein kinase A (PKA) activators (forskolin and 8‐Br‐cAMP) stimulated α‐methyl D ‐glucopyranoside uptake. Kinetic analysis revealed an increase in both V_max and affinity of the transport. Immunohistochemistry and biotinylation experiments showed that this stimulation was accompanied by an increased amount of SGLT1 localized into the plasma membrane, which explains the higher V_max of the transport. Cytochalasin D only partly attenuated the effect of forskolin as did deletion of the PKA phosphorylation site of SGLT1 in transient transfection studies. Experiments using an anti‐phosphopeptide antibody revealed that forskolin also increased the extent of phosphorylation of SGLT1 in the membrane fraction. These results suggested that regulation of SGLT1 mediated glucose transport involves an additional direct effect on SGLT1 by phosphorylation. To evaluate this assumption further, phosphorylation studies of recombinant human SGLT1 (hSGLT1) in vitro were performed. In the presence of the catalytic subunit PKA and [³²P] ATP 1.05 mol of phosphate were incorporated/mol of hSGLT1. Additionally, phosphorylated hSGLT1 demonstrated a reduction in tryptophan fluorescence intensity and a higher quenching by the hydrophilic Trp quencher acrylamide, particularly in the presence of D ‐glucose. These results indicate that PKA‐mediated phosphorylation of SGLT1 changes the conformation of the empty carrier and the glucose carrier complex, probably causing the increase in transport affinity. Thus, PKA‐mediated phosphorylation of the transporter represents a further mechanism in the regulation of SGLT1‐mediated glucose transport in epithelial cells, in addition to a change in surface membrane expression. J. Cell. Biochem. 106: 444–452, 2009. © 2008 Wiley‐Liss, Inc.     

A missense mutation in the Na(+)/glucose cotransporter gene SGLT1 in a patient with congenital glucose-galactose malabsorption: normal trafficking but inactivation of the mutant protein

The Na(+)/glucose cotransporter gene SGLT1 was analyzed in a Japanese patient with congenital glucose-galactose malabsorption. Genomic DNA was used as a template for amplification by the polymerase chain reaction of each of the 15 exons of SGLT1. The amplification products were cloned and sequenced. About half of the exon 5 clones of the patient contained a C-->T transition, resulting in an Arg(135)-->Trp mutation, whereas the remaining clones contained the normal exon 5 sequence. In addition, whereas some exon 12 clones exhibited the normal sequence, others showed a CAgtaggtatcatc-->CAgacc mutation at the splice donor site of intron 12 that may result either in the skipping of exon 12 or in read-through of intron 12. Neither the Arg(135)-->Trp mutant nor either of the possible intron 12 mutant proteins exhibited Na(+)-dependent glucose transport activity when expressed in Xenopus oocytes. Immunocytochemical analysis indicated, however, that the Arg(135)-->Trp mutant was localized to the oocyte plasma membrane. DNA sequence analysis revealed that the missense mutation in exon 5 and the splice site mutation in intron 12 were inherited from the proband's father and mother, respectively. These results indicate that the patient is a compound heterozygote for this disease, and that the Arg(135)-->Trp mutant of SGLT1 undergoes normal trafficking to the plasma membrane but is non-functional.     

Sugar binding residue affects apparent Na+ affinity and transport stoichiometry in mouse sodium/glucose cotransporter type 3B

SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.     

Mouse SGLT3a generates proton-activated currents but does not transport sugar

Sodium-glucose cotransporters (SGLTs) are secondary active transporters belonging to the SLC5 gene family. SGLT1, a well-characterized member of this family, electrogenically transports glucose and galactose. Human SGLT3 (hSGLT3), despite sharing a high amino acid identity with human SGLT1 (hSGLT1), does not transport sugar, although functions as a sugar sensor. In contrast to humans, two different genes in mice and rats code for two different SGLT3 proteins, SGLT3a and SGLT3b. We previously cloned and characterized mouse SGLT3b (mSGLT3b) and showed that, while it does transport sugar like SGLT1, it likely functions as a physiological sugar sensor like hSGLT3. In this study, we cloned mouse SGLT3a (mSGLT3a) and characterized it by expressing it in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. mSGLT3a did not transport sugar, and sugars did not induce currents at pH 7.4, though acidic pH induced inward currents that increased in the presence of sugar. Moreover, mutation of residue 457 from glutamate to glutamine resulted in a Na(+)-dependent transport of sugar that was inhibited by phlorizin. To corroborate our results in oocytes, we expressed and characterized mSGLT3a in mammalian cells and confirmed our findings. In addition, we cloned, expressed, and characterized rat SGLT3a in oocytes and found characteristics similar to mSGLT3a. In summary, acidic pH induces currents in mSGLT3a, and sugar-induced currents are increased at acidic pH, but wild-type SGLT3a does not transport sugar.     

Structural Insights into Genetic Variants of Na+/Glucose Cotransporter SGLT1 Causing Glucose–Galactose Malabsorption: vSGLT as a Model Structure

Current advances in structural biology provide valuable insights into structure-function relationship of membrane transporters by solving crystal structures of bacterial homologs of human transporters. Therefore, scientists consider bacterial transporters as useful structural models for designing of drugs targeted in human diseases. The functional homology between Vibrio parahaemolyticus Na(+)/galactose transporter (vSGLT) and Na(+)/glucose cotransporter SGLT1 has been well established a decade ago. Now the crystal structure of vSGLT is considered quite valuable in explaining not only the cotransport mechanisms, but it also acts as a representative protein in understanding the protein stability and amino acid interactions within the core structure. We investigated the molecular mechanisms of genetic variations in SGLT1 that cause glucose-galactose malabsorption (GGM) defects using the crystal structure of vSGLT as a model sugar transporter. Our in silico mutagenesis and modeling analysis suggest that the GGM genetic variations lead to conformational changes either by structure destabilization or by formation of unnecessary interaction within the core structure of SGLT1 thereby explaining the genetic defects in Na(+) dependent sugar translocation across the cell membrane.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Kinetics of the Reverse Mode of the Na<Superscript>+</Superscript>/Glucose Cotransporter

This study investigates the reverse mode of the Na⁺/glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na⁺- and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The current-voltage relationship saturated at positive membrane voltages (30–50 mV), and approached zero at −150 mV. The half-maximal concentration for αMDG-evoked outward current (K_0.5^αMDG) was 35 mM (at 0 mV). In comparison, K_0.5^αMDG for forward sugar transport was 0.15 mM (at 0 mV). K_0.5^Na was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-O-methyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na⁺/sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.     

Sodium-independent low-affinity D-glucose transport by human sodium/D-glucose cotransporter 1: critical role of tryptophan 561

Although there is no evidence of significant Na-independent glucose flux in tissues naturally expressing SGLT1, previous kinetic and biophysical studies suggest that sodium/d-glucose cotransporter 1 (hSGLT1) can facilitate sodium-independent d-glucose transport and may contain more than one sugar binding site. In this work, we analyze the kinetic properties and conformational states of isolated hSGLT1 reconstituted in liposomes by transport and fluorescence studies in the absence of sodium. In the transport studies with hSGLT1, significant sodium-independent phlorizin inhibitable alpha-methyl d-glucopyranoside (alpha-MDG) uptake was observed which amounted to approximately 20% of the uptake observed in the presence of a sodium gradient. The apparent affinity constant for alpha-MDG was thereby 3.4 +/- 0.5 mM, a value approximately 10-fold higher than that in the presence of sodium. In the absence of sodium, various sugars significantly decreased the intrinsic Trp fluorescence of hSGLT1 in proteoliposomes exhibiting the following sequence of affinities: alpha-MDG > d-glucose approximately d-galactose > 6-deoxy-d-glucose > 2-deoxy-d-glucose > d-allose. Furthermore, significant protection effects of d-glucose or phlorizin against potassium iodide, acrylamide, or trichloroethanol quenching were observed. To locate the Trps involved in this reaction, we generated mutants in which all Trps were sequentially substituted with Phe. None of the replacements significantly affected sodium-dependent uptake. Uptake in the absence of sodium and typical fluorescence changes depended, however, on the presence of Trp at position 561. This Trp residue is conserved in all known SGLT1 forms (except Vibrio parahaemolyticus SGLT) and all SGLT isoforms in humans (except hSGLT3). If all these data are taken into consideration, it seems that Trp-561 in hSGLT1 forms part of a low-affinity sodium-independent binding and/or translocation site for d-glucose. The rate of sodium-independent translocation via hSGLT1 seems, however, to be tightly regulated in the intact cell by yet unknown factors.     

Identification of NF2 germ-line mutations and comparison with neurofibromatosis 2 phenotypes

Neurofibromatosis 2 (NF2) is an autosomal inherited disorder that predisposes carriers to nervous system tumors. To examine genotype-phenotype correlations in NF2, we performed mutation analyses and gadolinium-enhanced magnetic resonance imaging of the head and full spine in 59 unrelated NF2 patients. In patients with vestibular schwannomas (VSs) or identified NF2 mutations, the mild phenotype was defined as <2 other intracranial tumors and ≤ 4 spinal tumors, and the severe phenotype as either ≥ 2 other intracranial tumors or > 4 spinal tumors. Nineteen mutations were found in 20 (34%) of the patients and were distributed in 12 of the 17 exons of the NF2 gene, including intron-exon boundaries. Seven mutations were frameshift, six were nonsense, four were splice site, two were missense, and one was a 3-bp in frame deletion. The nonsense mutations included one codon 57 and two codon 262 C→T transitions in CpG dinucleotides. The frameshift and nonsense NF2 mutations occurred primarily in patients with severe phenotypes. The two missense mutations occurred in patients with mild phenotypes, and three of the four splice site mutations occurred in families with both mild and severe phenotypes. Truncating NF2 mutations are usually associated with severe phenotypes, but the association of some mutations with mild and severe phenotypes indicates that NF2 expression is influenced by stochastic, epigenetic, or environmental factors. Received: 4 July 1996     

Regulation of Na(+)-coupled glucose carrier SGLT1 by human papillomavirus 18 E6 protein

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na⁺-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I_g). I_g was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I_g following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1.     

Regulation of the human Na⁺-dependent glucose cotransporter hSGLT2

The human Na(+)-glucose cotransporter SGLT2 is expressed mainly in the kidney proximal convoluted tubule where it is considered to be responsible for the bulk of glucose reabsorption. Phosphorylation profiling has revealed that SGLT2 exists in a phosphorylated state in the rat renal proximal tubule cortex, so we decided to investigate the regulation of human SGLT2 (hSGLT2) by protein kinases. hSGLT2 was expressed in human embryonic kidney (HEK) 293T cells, and the activity of the protein was measured using radiotracer and whole cell patch-clamp electrophysiology assays before and after activation of protein kinases. 8-Bromo-adenosine cAMP (8-Br-cAMP) was used to activate protein kinase A, and sn-1,2-dioctanoylglycerol (DOG) was used to activate protein kinase C (PKC). 8-Br-cAMP stimulated D-[α-methyl-(14)C]glucopyranoside ([(14)C]α-MDG) uptake and Na(+)-glucose currents by 200% and DOG increased [(14)C]α-MDG uptake and Na(+)-glucose currents by 50%. In both cases the increase in SGLT2 activity was marked by an increase in the maximum rate of transport with no change in glucose affinity. These effects were completely negated by mutation of serine 624 to alanine. Insulin induced a 250% increase in Na(+)-glucose transport by wild-type but not S624A SGLT2. Parallel studies confirmed that the activity of hSGLT1 was regulated by PKA and PKC due to changes in the number of transporters in the cell membrane. hSGLT1 was relatively insensitive to insulin. We conclude that hSGLT1 and hSGLT2 are regulated by different mechanisms and suggest that insulin is an SGLT2 agonist in vivo.     

Structural selectivity of human SGLT inhibitors

Human Na(+)-D-glucose cotransporter (hSGLT) inhibitors constitute the newest class of diabetes drugs, blocking up to 50% of renal glucose reabsorption in vivo. These drugs have potential for widespread use in the diabetes epidemic, but how they work at a molecular level is poorly understood. Here, we use electrophysiological methods to assess how they block Na(+)-D-glucose cotransporter SGLT1 and SGLT2 expressed in human embryonic kidney 293T (HEK-293T) cells and compared them to the classic SGLT inhibitor phlorizin. Dapagliflozin [(1S)-1,5,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucitol], two structural analogs, and the aglycones of phlorizin and dapagliflozin were investigated in detail. Dapagliflozin and fluoro-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-4-F-4-deoxy-D-glucitol] blocked glucose transport and glucose-coupled currents with ≈100-fold specificity for hSGLT2 (K(i) = 6 nM) over hSGLT1 (K(i) = 400 nM). As galactose is a poor substrate for SGLT2, it was surprising that galacto-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-galactitol] was a selective inhibitor of hSGLT2, but was less potent than dapagliflozin for both transporters (hSGLT2 K(i) = 25 nM, hSGLT1 K(i) = 25,000 nM). Phlorizin and galacto-dapagliflozin rapidly dissociated from SGLT2 [half-time off rate (t(1/2,Off)) ≈ 20-30 s], while dapagliflozin and fluoro-dapagliflozin dissociated from hSGLT2 at a rate 10-fold slower (t(1/2,Off) ≥ 180 s). Phlorizin was unable to exchange with dapagliflozin bound to hSGLT2. In contrast, dapagliflozin, fluoro-dapagliflozin, and galacto-dapagliflozin dissociated quickly from hSGLT1 (t(1/2,Off) = 1-2 s), and phlorizin readily exchanged with dapagliflozin bound to hSGLT1. The aglycones of phlorizin and dapagliflozin were poor inhibitors of both hSGLT2 and hSGLT1 with K(i) values > 100 μM. These results show that inhibitor binding to SGLTs is composed of two synergistic forces: sugar binding to the glucose site, which is not rigid, and so different sugars will change the orientation of the aglycone in the access vestibule; and the binding of the aglycone affects the binding affinity of the entire inhibitor. Therefore, the pharmacophore must include variations in both the structure of the sugar and the aglycone.     

Glycoside Binding and Translocation in Na+-Dependent Glucose Cotransporters: Comparison of SGLT1 and SGLT3

Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na⁺/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K_0.5 0.06 mm) and 2-naphthylglucose with lower affinity (K_0.5 0.5 mm) than α-methyl-d-glucopyranoside (αMDG, 0.2 mm). Both were poorly transported (maximal velocities, I _max , 14% and 8% of αMDG). Other compounds were inhibitors (K _i s 1–13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than αMDG (K_0.5s 0.9, 0.2 and 2.5 mm and relative I _max s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I _max s (130 and 120%) and comparable K_0.5s (8 and 1 mm). Increased affinity of indican relative to αMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value. Received: 18 February 2000/Revised: 13 April 2000     

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

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°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 V _max 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 V _max 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 (K _i = 15 μm). 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 K _m 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. Received: 25 October 1995/Revised: 10 June 1996     

Determination of the Na(+)/glucose cotransporter (SGLT1) turnover rate using the ion-trap technique

The Na⁺/glucose cotransporter (SGLT1) is a membrane protein that couples the transport of two Na⁺ ions and one glucose molecule using the so-called alternating access mechanism. According to this principle, each cotransporter molecule can adopt either of two main conformations: one with the binding sites accessible to the extracellular solution and one with the binding sites facing the intracellular solution. The turnover rate (TOR) is the number of complete cycles that each protein performs per second. Determination of the TOR has important consequences for investigation of the cotransport mechanism, as none of the rate constants involved in mediating transport in a given direction (conformational changes and binding and unbinding reactions) can be slower than the TOR measured under the same conditions. In addition, the TOR can be used to estimate the number of cotransporter molecules involved in generating a given ensemble activity. In this study, we obtain an independent estimation of the TOR for human SGLT1 expressed in Xenopus laevis oocytes applying the ion-trap technique. This approach detects the quantity of ions released in or taken up from the restricted space existing between the oocyte plasma membrane and the tip of a large ion-selective electrode. Taking advantage of the fact that hSGLT1 in the absence of Na⁺ can cotransport glucose with protons, we used a pH electrode to determine a TOR of 8.00 ± 1.3 s⁻¹ in the presence of 35 mM α-methyl-glucose at −150 mV (pH 5.5). For the same group of oocytes, a TOR of 13.3 ± 2.4 s⁻¹ was estimated under near-V_max conditions, i.e., in the presence of 90 mM Na⁺ and 5 mM α-methyl-glucose. Under these circumstances, the average cotransport current was −1.08 ± 0.61 μA (n = 14), and this activity was generated by an average of 3.6 ± 0.7 × 10¹¹ cotransporter molecules/oocyte.