Bruce A. Hirayama

Biology of Human Sodium Glucose Transporters

There are two classes of glucose transporters involved in glucose homeostasis in the body, the facilitated transporters or uniporters (GLUTs) and the active transporters or symporters (SGLTs). The energy for active glucose transport is provided by the sodium gradient across the cell membrane, the Na(+) glucose cotransport hypothesis first proposed in 1960 by Crane. Since the cloning of SGLT1 in 1987, there have been advances in the genetics, molecular biology, biochemistry, biophysics, and structure of SGLTs. There are 12 members of the human SGLT (SLC5) gene family, including cotransporters for sugars, anions, vitamins, and short-chain fatty acids. Here we give a personal review of these advances. The SGLTs belong to a structural class of membrane proteins from unrelated gene families of antiporters and Na(+) and H(+) symporters. This class shares a common atomic architecture and a common transport mechanism. SGLTs also function as water and urea channels, glucose sensors, and coupled-water and urea transporters. We also discuss the physiology and pathophysiology of SGLTs, e.g., glucose galactose malabsorption and familial renal glycosuria, and briefly report on targeting of SGLTs for new therapies for diabetes.

Active sugar transport in health and disease

Secondary active glucose transport occurs by at least four members of the SLC5 gene family. This review considers the structure and function of two premier members, SGLT1 and SGLT2, and their role in intestinal glucose absorption and renal glucose reabsorption. Genetics disorders of SGLTs include Glucose‐Galactose Malabsorption, and Familial Renal Glucosuria. SGLT1 plays a central role in Oral Rehydration Therapy used so effectively to treat secretory diarrhoea such as cholera. Increasing attention is being focused on SGLTs as drug targets for the therapy of diabetes.

The Crystal Structure of a Sodium Galactose Transporter Reveals Mechanistic Insights into Na+/Sugar Symport

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The ∼3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

A glucose sensor hiding in a family of transporters

We have examined the expression and function of a previously undescribed human member (SGLT3/SLC5A4) of the sodium/glucose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na+-dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na+/glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine

SummaryThe Na+/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na+/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of14C-α-methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na+-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (Ki 6 and 9 μm), and saturable (α-methyl-d-glucopyranosideKm 110 and 570 μm). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>α-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na+/glucose cotransporter.

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s⁻¹) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.

Passive water and ion transport by cotransporters.

1 The rabbit Na+‐glucose (SGLT1) and the human Na+‐Cl−‐GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker‐sensitive Na+ currents in the absence of substrates (glucose and GABA). 3 Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3‐5 μM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 μM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 4 The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol−1, respectively. The high Ea for Na+ transport is comparable to that of Na+‐glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 5 GAT1 also behaved as an SKF89976A‐sensitive water channel. We did not observe passive Na+ transport through GAT1. 6 We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.

Distribution of the SGLT1 Na+glucose cotransporter and mRNA along the crypt-villus axis of rabbit small intestine

The expression of the Na+/glucose cotransporter (SGLT1) mRNA and protein along the crypt-villus axis of the rabbit small intestine was examined using in situ hybridization and immunocytochemical techniques. We detected mRNA in the cells on the villus, but not in the crypts, and the mRNA abundance increased 6-fold from the base to the tip of the villus. SGLT1 protein was restricted to the brush borders of mature enterocytes. We suggest that the high rate of sugar transport across the tips of the villus is due to the transcription of the SGLT1 gene in mature enterocytes, the subsequent translation of SGLT mRNA, and the insertion direct of the functional SGLT1 transporter into the brush border membrane of these cells lining the villus tip.

Phenylglucosides and the Na+/glucose cotransporter (SGLT1): analysis of interactions.

Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na+-dependent uptake of 14C-α-methyl-d-glucopyranoside (αMDG) with apparent Kis in the range 0.1 to 20 mm, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na+-loaded transporter. Computational chemistry indicated that all these β-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the-NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.

Cation Effects on Protein Conformation and Transport in the Na+/Glucose Cotransporter

Cation-driven cotransporters are essential membrane proteins in procaryotes and eucaryotes, which use the energy of the transmembrane electrochemical gradient to drive transport of a substrate against its concentration gradient. Do they share a common mechanism? Cation selectivity of the rabbit isoform of the Na+/glucose cotransporter (SGLT1) was examined using the twoelectrode voltage clamp and the Xenopus oocyte expression system. The effect of H+, Li+, and Na+ on kinetics of SGLT1 was compared to the effects of these cations on the bacterial melibiose. In SGLT1, substitution of H+ or Li+ for Na+ caused a kinetic penalty in that the apparent affinity for sugar (K0.5sugar) decreased by an order of magnitude or more (from 0.2 to 30 mM) depending on the membrane potential and cation. The effect of the cation on the K0.5sugar/V profiles was independent of the sugar for glucose and α-methyl-β-D-glucose; this profile was maintained for galactose in Li+ and Na+, but was 2 orders of magnitude higher in H+, but the Imax for glucose, galactose, and α-methyl-β-D-glucose in a given cation were identical. Li+ supported a lower maximal rate of transport (Imax) than Na+ (∼80% of ImaxNa), while the Imax in H+ was higher than Na+ (≥180% of ImaxNa). Our interpretation of these results and simulations using a six-state mathematical model, are as follows. 1) Binding of the cation causes a conformational change in the sugar binding pocket, the exact conformation being determined by the specific cation. 2) Once the sugar is bound, it is transported at a characteristic rate determined by the cation. 3) Mathematical simulations suggest that the largest contribution to the kinetic variability of both cation and sugar transport is associated with cation binding. Similarity to the effects of cation substitution in MelB suggests that the mechanism of energy coupling has been evolutionarily conserved.