Mechanistic Insights into Protein Stability and Self-aggregation in GLUT1 Genetic Variants Causing GLUT1-Deficiency Syndrome.

Human sodium-independent glucose cotransporter 1 (hGLUT1) has been studied for its tetramerization and multimerization at the cell surface. Homozygous or compound heterozygous mutations in hGLUT1 elicit GLUT1-deficiency syndrome (GLUT1-DS), a metabolic disorder, which results in impaired glucose transport into the brain. The reduced cell surface expression or loss of function have been shown for some GLUT1 mutants. However, the mechanism by which deleterious mutations affect protein structure, conformational stability and GLUT1 oligomerization is not known and require investigation. In this review, we combined previous knowledge of GLUT1 mutations with hGLUT1 crystal structure to analyze native interactions and several natural single-point mutations. The modeling of native hGLUT1 structure confirmed the roles of native residues in forming a range of side-chain interactions. Interestingly, the modeled mutants pointed to the formation of a variety of non-native novel interactions, altering interaction networks and potentially eliciting protein misfolding. Self-aggregation of the last part of hGLUT1 was predicted using protein aggregation prediction tool. Furthermore, an increase in aggregation potential in the aggregation-prone regions was estimated for several mutants suggesting increased aggregation of misfolded protein. Protein stability change analysis predicted that GLUT1 mutant proteins are unstable. Combining GLUT1 oligomerization behavior with our modeling, aggregation prediction, and protein stability analyses, this work provides state-of-the-art view of GLUT1 genetic mutations that could destabilize native interactions, generate novel interactions, trigger protein misfolding, and enhance protein aggregation in a disease state.

Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl ß-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

A biophysical glance at the outer surface of the membrane transporter SGLT1.

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure-function relation. Crystallographic studies, if available, define the transmembrane segments in a "frozen" state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein. The current review presents biophysical methods that can shed light on the dynamics of transporters in the membrane. The techniques that are presented in some detail are single-molecule recognition atomic force microscopy and tryptophan scanning, which can report on the positioning of the loops and on conformational changes at the outer surface. Studies on a variety of symporters are discussed, which use gradients of sodium or protons as energy source to translocate (mainly organic) solutes against their concentration gradients into or out of the cells. Primarily, investigations of the sodium-glucose cotransporter SGLT1 are used as examples for this biophysical approach to understand transporter function.

SGLT inhibitors as new therapeutic tools in the treatment of diabetes.

Recently, the idea has been developed to lower blood glucose blood glucose levels in diabetes by inhibiting sugar reabsorption sugar reabsorption in the kidney kidney . The main target is thereby the early proximal tubule proximal tubule where secondary active transport secondary active transport of the sugar is mediated by the sodium-D: -glucose D-glucose cotransporter SGLT2 SGLT2 . A model substance for the inhibitors inhibitors is the O-glucoside O-glucoside phlorizin phlorizin which inhibits transport transport competitively. Its binding to the transporter involves at least two different domains: an aglucone binding aglucone binding site at the transporter surface, involving extramembranous loops extramembraneous loops , and the sugar binding sugar binding /translocation site buried in a hydrophilic pocket of the transporter. The properties of these binding sites differ between SGLT2 and SGLT1 SGLT1 , which mediates sugar absorption sugar absorption in the intestine intestine . Various O-, C-, N- and S-glucosides have been synthesized with high affinity affinity and high specificity specificity for SGLT2 SGLT2 . Some of these glucosides are in clinical trials clinical trials and have been proven to successfully increase urinary glucose excretion urinary glucose excretion and to decrease blood sugar blood sugar levels without the danger of hypoglycaemia hypoglycaemia during fasting fasting in type 2 diabetes type 2 diabetes .

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 alpha-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 [(32)P] 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.

A proteomic study of sodium/D-glucose cotransporter 1 (SGLT1): topology of loop 13 and coverage of other functionally important domains.

In order to obtain further information about the structure and function of human sodium/D-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate. Sequence coverage was provided from amino acids 559 to 644, suggesting that loop 13 is almost completely accessible at the extravesicular face of the proteoliposomes. These results support the notion that major parts of loop 13, essential for the interaction with transport inhibitors in vivo, are located extracellularly in intact cells. In-gel trypsin, chymotrypsin, and in particular trypsin/chymotrypsin digestion of recombinant SGLT1 in combination with LC-MS/MS provide extensive sequence coverage of the protein, including domains involved in sugar and inhibitor binding and potential phosphorylation sites. These studies demonstrate that proteomic analysis combined with mass spectrometry is a useful tool to characterize regions of SGLT1 that are important for its function and regulation.

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/D-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence.

Recombinant purified human sodium/D-glucose cotransporter1 (hSGLT1) was reconstituted in a functional form into phospholipid vesicles and its conformational states in the absence and presence of ligands and inhibitors were probed by intrinsic tryptophan fluorescence. In the presence of sodium, sugars increase intrinsic fluorescence (maximum 17%) in a saturable manner in the following order alpha-MDG >D-Glu approximately D-Gal >> D-Man >D-All, with no effect of L-Glu. Apparent affinities ranging from 0.65 to 10.4 mM were observed. In addition, D-Glu increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary, the transport inhibitor phlorizin decreased Trps fluorescence in a sodium-dependent manner by 50% with a red shift of 4-6 nm and decreased quencher accessibility, these effects were saturable with a high affinity of 5 microM. Furthermore, the positioning of the tryptophans in the reconstituted transporter was investigated. hSGLT1 Trps fluorescence was reduced by N-bromosuccinimide treatment maximally 25% in membranes and 65% in solution. The fluorescence was also significantly but differently quenched by the lipid-soluble spin labeled probes 5-Doxyl-phosphatidylcholine (40%) and 12-Doxyl-phosphatidylcholine (26%). Depth-calculation using the parallax method suggested a location of Trps at an average depth of 10 angstrom from the center of the bilayer. These studies demonstrate the existence of different conformational states of the membrane-embedded transporter in its glucose-free form, as sodium-glucose-carrier complex and as sodium-phlorizin-carrier complex. They further indicate that most of the Trp residues in hSGLT1 are located in hydrophobic regions of the protein or in contact with the lipid bilayer of the membrane. There, they are located close to the membrane-water interface contributing to the vectorial nature of the transporter.

Thioglycosides as inhibitors of hSGLT1 and hSGLT2: potential therapeutic agents for the control of hyperglycemia in diabetes.

The treatment of diabetes has been mainly focused on maintaining normal blood glucose concentrations. Insulin and hypoglycemic agents have been used as standard therapeutic strategies. However, these are characterized by limited efficacy and adverse side effects, making the development of new therapeutic alternatives mandatory. Inhibition of glucose reabsorption in the kidney, mediated by SGLT1 or SGLT2, represents a promising therapeutic approach. Therefore, the aim of the present study was to evaluate the effect of thioglycosides on human SGLT1 and SGLT2. For this purpose, stably transfected Chinese hamster ovary (CHO) cells expressing human SGLT1 and SGLT2 were used. The inhibitory effect of thioglycosides was assessed in transport studies and membrane potential measurements, using alpha-methyl-glucoside uptake and fluorescence resonance energy transfer, respectively. We found that some thioglycosides inhibited hSGLT more strongly than phlorizin. Specifically, thioglycoside I (phenyl-1'-thio-beta-D-glucopyranoside) inhibited hSGLT2 stronger than hSGLT1 and to a larger extent than phlorizin. Thioglycoside VII (2-hydroxymethyl-phenyl-1'-thio-beta-D-galacto-pyranoside) had a pronounced inhibitory effect on hSGLT1 but not on hSGLT2. Kinetic studies confirmed the inhibitory effect of these thioglycosides on hSGLT1 or hSGLT2, demonstrating competitive inhibition as the mechanism of action. Therefore, these thioglycosides represent promising therapeutic agents for the control of hyperglycemia in patients with diabetes.

Sodium-phosphate cotransporter in human salivary glands: molecular evidence for the involvement of NPT2b in acinar phosphate secretion and ductal phosphate reabsorption.

OBJECTIVE: In order to elucidate the cellular and molecular mechanisms of phosphate secretion by human salivary glands, the expression and intracellular distribution of sodium-phosphate cotransporters was investigated. DESIGN: Total RNA was extracted from 33 parotid gland (PG) and 35 submandibular gland (SMG) samples and RT-PCR was performed using gene specific primers for all known sodium-phosphate cotransporters. An antibody was raised against an NPT2b epitope and the cellular and intracellular distribution was investigated by immunohistochemistry. RESULTS: No mRNA for the type I cotransporter NPT1 was found. Out of the type II phosphate cotransporters only message for NPT2b but not for NPT2a or NPT2c could be detected in about the same number of samples (76% in PG versus 69% in SMG). Type III cotransporter mRNA was also found in both glands, PIT1 gave positive results for 93% of PG samples compared to 69% of SMG samples. For PIT2 also, a higher expression was found in PG than in SMG, although the difference was smaller (79% versus 51%). Immunostaining for NPT2b was found both in the acini and in the ducts, with a stronger reaction in the latter. In acinar cells, NPT2b was restricted to the basal-lateral plasma membrane, in duct cells, a broad band of reactivity was located in the apical part of the cell. CONCLUSIONS: These findings suggest a secondary active secretion of phosphate into the primary saliva. Ductal cells appear to be able to reabsorb phosphate, thereby modifying the phosphate concentration in the final saliva.

Pathogenic mutations causing glucose transport defects in GLUT1 transporter: The role of intermolecular forces in protein structure-function.

Two families of glucose transporter - the Na(+)-dependent glucose cotransporter-1 (SGLT family) and the facilitated diffusion glucose transporter family (GLUT family) - play a crucial role in the translocation of glucose across the epithelial cell membrane. How genetic mutations cause life-threatening diseases like GLUT1-deficiency syndrome (GLUT1-DS) is not well understood. In this review, we have combined previous functional data with our in silico analyses of the bacterial homologue of GLUT members, XylE (an outward-facing, partly occluded conformation) and previously proposed GLUT1 homology model (an inward-facing conformation). A variety of native and mutant side chain interactions were modeled to highlight the potential roles of mutations in destabilizing protein-protein interaction hence triggering structural and functional defects. This study sets the stage for future studies of the structural properties that mediate GLUT1 dysfunction and further suggests that both SGLT and GLUT families share conserved domains that stabilize the transporter structure/function via a similar mechanism.

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system.

The sodium glucose cotransporter SGLT1 expressed mainly in the intestine and kidney has been explored extensively for understanding the mechanism of sugar cotransport and its inhibition by a classical competitive inhibitor, phlorizin (Pz). It has been shown that inhibition of SGLT1 by Pz involves its interaction followed by major conformational changes in the Pz binding domain (PBD) in C-terminal loop 13. However, the mechanism of Pz inhibition and its interaction with other members of SGLT is not known. In this hypothesis, we performed molecular modeling of SGLT1-loop 13 with Pz and carried out primary sequence analyses and secondary structure predictions to determine qualitatively similar PBDs in C-termini of human SGLT2-4, except for vSGLT, which contains an unstructured short C-terminus. The ranking of predictions of Pz interaction strongly agrees with the following ranking of previously reported Pz inhibition: SGLT2>SGLT1>SGLT4>SGLT3>>vSGLT. In addition, the sugar binding residues were found to be quite conserved among all SGLT members investigated here. Based on these preliminary analyses, we propose that other Pz-sensitive SGLTs are also inhibited via mechanism similar to SGLT1 where an aglucone of Pz, phloretin, interacts with PBD and glucoside moiety with sugar binding residues. Our hypothesis sets the stage for future analyses on investigation of Pz interaction with SGLT family and further suggests that Pz modeling may be explored to design novel inhibitors targeting several SGLT members.

Ethanol treatment of hepatocellular carcinoma: high potentials of low concentrations.

Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, which is associated with a very poor prognosis. A curative treatment is difficult to achieve and is only possible in a low number of patients. Therefore, many different therapeutic strategies have been developed as alternative treatment. Among these, percutaneous injection of high concentrations of ethanol (>50 mM) has been proven to be effective for the treatment of small HCC (less than 3 cm in diameter). However, the principal problem with using ethanol is its toxic effects on non-tumor cells adjacent to the tumor area. The objective of this review is to juxtapose the therapeutic potential of high and low concentrations of ethanol in the treatment of HCC, based on experimental studies obtained with the human hepatocellular tumor cell line (HepG2). They have shown that high concentrations of ethanol lead to necrosis, while low concentrations induce apoptosis due to activation of Fas-receptors. Triggering of apoptosis through Fas-receptors represents a mechanism of action different from that observed with high concentrations of ethanol, thus, reducing the complications that follow the inflammatory process due to necrosis. Therefore, the use of low concentrations of ethanol could be an effective treatment for HCC.

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.

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.

Single-molecule recognition force spectroscopy of transmembrane transporters on living cells.

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.

C-terminal loop 13 of Na+/glucose cotransporter 1 contains both stereospecific and non-stereospecific sugar interaction sites.

To investigate whether the C-terminal loop 13 of rabbit sodium/glucose cotransporter SGLT1 is involved in the recognition of the substrate d-glucose, isolated loop 13 (amino acids (aa) 541-638) was immobilized to a lipid bilayer. Interactions were investigated by surface plasmon resonance spectroscopy using an antibody directed against the late part of the loop (aa 606-631) or the glucoside transport inhibitor phlorizin. Specific binding of the antibody to the loop could be detected. The number of bound antibodies decreased upon the addition of d-glucose but not upon the addition of l-glucose. Phlorizin also significantly lowered the number of bound antibodies. Binding of phlorizin to the loop could also be demonstrated directly. Binding of phlorizin was, however, reduced to a similar extent upon the addition of either d-glucose or l-glucose, indicating their unspecific competition with the inhibitor's sugar moiety. Thus, the presence of a stereospecific glucose interaction site in the late part of the loop and a second, but non-stereospecific, sugar binding site on the same loop was assumed. To investigate whether the early part of loop 13 contains this non-stereospecific sugar binding site, peptides containing aa 541-598 were expressed in Escherichia coli and purified. Both d-glucose and l-glucose quenched the peptides tryptophan fluorescence and reduced the Trp accessibility to acrylamide to a similar degree. In view of the recently proposed transmembrane orientation of loop 13, the two binding sites may be part of the extracellular (stereospecific) and intracellular (non-stereospecific) sugar interaction sites of SGLT1.

Three surface subdomains form the vestibule of the Na+/glucose cotransporter SGLT1.

A combination of biophysical and biochemical approaches was employed to probe the topology, arrangement, and function of the large surface subdomains of SGLT1 in living cells. Using atomic force microscopy on the single molecule level, Chinese hamster ovary cells overexpressing SGLT1 were probed with atomic force microscopy tips carrying antibodies against epitopes of different subdomains. Specific single molecule recognition events were observed with antibodies against loop 6-7, loop 8-9, and loop 13-14, demonstrating the extracellular orientation of these subdomains. The addition of D-glucose in Na+-containing medium decreased the binding probability of the loop 8-9 antibody, suggesting a transport-related conformational change in the region between amino acids 339 and 356. Transport studies with mutants C345A, C351A, C355A, or C361S supported a role for these amino acids in determining the affinity of SGLT1 for D-glucose. MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate and dithiothreitol inhibition patterns on alpha-methyl-glucoside uptake by COS-7 cells expressing C255A, C560A, or C608A suggested the presence of a disulfide bridge between Cys255 and Cys608. This assumption was corroborated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry showing mass differences in peptides derived from transporters biotinylated in the absence and presence of dithiothreitol. These results indicate that loop 6-7 and loop 13-14 are connected by a disulfide bridge. This bridge brings also loop 8-9 into close vicinity with the former subdomains to create a vestibule for sugar binding.

Differential regulation of cell volume and shape in confluent rat hepatocytes under hypertonic stress.

In confluent primary cultures of rat hepatocytes,hypertonic stress leads to cell shrinkage and activates non-selective cation channels as the main mechanism of regulatory cell volume increase. The process is found to employ the exocytotic insertion of channels into the plasma membrane and (in addition to PKC) PLC, tyrosine kinases and G proteins, but not PI 3-kinase are part of the signalling network. Furthermore, hypertonic stress leads to the formation of stress fibres and significantly alters the activity of RhoA, Rac and Cdc42. These latter effects, however, are likely to reflect the restoration of cell shape rather than the regulation of cell volume, both most probably converging at the level of focal adhesions and integrins.

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies.

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.

Na+ -D-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution.

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.