Characterization of the transport activity of SGLT2/MAP17, the renal low-affinity Na+-glucose cotransporter.

The cotransporter SGLT2 is responsible for 90% of renal glucose reabsorption, and we recently showed that MAP17 appears to work as a required ß-subunit. We report in the present study a detailed functional characterization of human SGLT2 in coexpression with human MAP17 in Xenopus laevis oocytes. Addition of external glucose generates a large inward current in the presence of Na, confirming an electrogenic transport mechanism. At a membrane potential of -50 mV, SGLT2 affinity constants for glucose and Na are 3.4 ± 0.4 and 18 ± 6 mM, respectively. The change in the reversal potential of the cotransport current as a function of external glucose concentration clearly confirms a 1:1 Na-to-glucose transport stoichiometry. SGLT2 is selective for glucose and α-methylglucose but also transports, to a lesser extent, galactose and 3-O-methylglucose. SGLT2 can be inhibited in a competitive manner by phlorizin (Ki = 31 ± 4 nM) and by dapagliflozin (Ki = 0.75 ± 0.3 nM). Similarly to SGLT1, SGLT2 can be activated by Na, Li, and protons. Pre-steady-state currents for SGLT2 do exist but are small in amplitude and relatively fast (a time constant of ~2 ms). The leak current defined as the phlorizin-sensitive current in the absence of substrate was extremely small in the case of SGLT2. In summary, in comparison with SGLT1, SGLT2 has a lower affinity for glucose, a transport stoichiometry of 1:1, very small pre-steady-state and leak currents, a 10-fold higher affinity for phlorizin, and an affinity for dapagliflozin in the subnanomolar range.

MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2.

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.

The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop.

Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence "quencher") to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12-13 loop is located on the external side of the membrane. As the 12-13 loop begins on the intracellular side of the membrane, this suggests that the 12-13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12-13 loop is re-entrant and readily accessible from the extracellular milieu.

The actual ionic nature of the leak current through the Na+/glucose cotransporter SGLT1.

Expression of the Na(+)/glucose cotransporter SGLT1 in Xenopus oocytes is characterized by a phlorizin-sensitive leak current (in the absence of glucose) that was originally called a "Na(+) leak" and represents some 5-10% of the maximal Na(+)/glucose cotransport current. We analyzed the ionic nature of the leak current using a human SGLT1 mutant (C292A) displaying a threefold larger leak current while keeping a reversal potential (V(R)) of approximately -15 mV as observed for wt SGLT1. V(R) showed only a modest negative shift when extracellular Na(+) concentration ([Na(+)](o)) was lowered and it was completely insensitive to changes in extracellular Cl(-). When extracellular pH (pH(o)) was decreased from 7.5 to 6.5 and 5.5, V(R) shifted by +15 and +40 mV, respectively, indicating that protons may be the main charge carrier at low pH(o) but other ions must be involved at pH(o) 7.5. In the presence of 15 mM [Na(+)](o) (pH(o) = 7.5), addition of 75 mM of either Na(+), Li(+), Cs(+), or K(+) generated similar increases in the leak current amplitude. This observation, which was confirmed with wt SGLT1, indicates a separate pathway for the leak current with respect to the cotransport current. This means that, contrary to previous beliefs, the leak current cannot be accounted for by the translocation of the Na-loaded and glucose-free cotransporter. Using chemical modification and different SGLT1 mutants, a relationship was found between the cationic leak current and the passive water permeability suggesting that water and cations may share a common pathway through the cotransporter.

A loss-of-function mutation in NaPi-IIa and renal Fanconi's syndrome.

We describe two siblings from a consanguineous family with autosomal recessive Fanconi's syndrome and hypophosphatemic rickets. Genetic analysis revealed a homozygous in-frame duplication of 21 bp in SLC34A1, which encodes the renal sodium-inorganic phosphate cotransporter NaPi-IIa, as the causative mutation. Functional studies in Xenopus laevis oocytes and in opossum kidney cells indicated complete loss of function of the mutant NaPi-IIa, resulting from failure of the transporter to reach the plasma membrane. These findings show that disruption of the human NaPi-IIa profoundly impairs overall renal phosphate reabsorption and proximal-tubule function and provide evidence of the critical role of NaPi-IIa in human renal phosphate handling.

Anionic leak currents through the Na+/monocarboxylate cotransporter SMCT1.

SMCT1 is a Na-coupled cotransporter of short chain monocarboxylates, which is expressed in the apical membrane of diverse epithelia such as colon, renal cortex, and thyroid. We previously reported that SMCT1 cotransport was reduced by extracellular Cl(-) replacement with cyclamate(-) and that the protein exhibited an ostensible anionic leak current. In this paper, we have revisited the interaction between small monovalent anions and SMCT cotransport and leak currents. We found that the apparent Cl(-) dependence of cotransport was due to inhibition of this protein by the replacement anion cyclamate, whereas several other replacement anions function as substrates for SMCT1; a suitable replacement anion (MES(-)) was identified. The observed outward leak currents represented anionic influx and favored larger anions (NO(3)(-)>I(-)>Br(-)>Cl(-)); currents in excess of 1 muA (at +50 mV) could be observed and exhibited a quasilinear relationship with anion concentrations up to 100 mM. Application of 25 mM bicarbonate did not produce measurable leak currents. The leak current displayed outward rectification, which disappeared when external Na(+) was replaced by N-methyl-d-glucamine(+). More precisely, external Na(+) blocked the leak current in both directions, but its K(i) value rose rapidly when membrane potential became positive. Thus SMCT1 possesses a anionic leak current that becomes significant whenever external Na(+) concentration is reduced. The presence of this leak current may represent a second function for SMCT1 in addition to cotransporting short chain fatty acids, and future experiments will determine whether this function serves a physiological role in tissues where SMCT1 is expressed.

Characterization of the null murine sodium/myo-inositol cotransporter 1 (Smit1 or Slc5a3) phenotype: myo-inositol rescue is independent of expression of its cognate mitochondrial ribosomal protein subunit 6 (Mrps6) gene and of phosphatidylinositol levels in neonatal brain.

Ablation of the murine Slc5a3 gene results in severe myo-inositol (Ins) deficiency and congenital central apnea due to abnormal respiratory rhythmogenesis. The lethal knockout phenotype may be rescued by supplementing the maternal drinking water with 1% Ins. In order to test the hypothesis that Ins deficiency leads to inositide deficiencies, which are corrected by prenatal treatment, we measured the effects of Ins rescue on Ins, phosphatidylinositol (PtdIns) and myo-inositol polyphosphate levels in brains of E18.5 knockout fetuses. As the Slc5a3 gene structure is unique in the sodium/solute cotransporter (SLC5) family, and exon 1 is shared with the mitochondrial ribosomal protein subunit 6 (Mrps6) gene, we also sought to determine whether expression of its cognate Mrps6 gene is abnormal in knockout fetuses. The mean level of Ins was increased by 92% in brains of rescued Slc5a3 knockout fetuses (0.48 versus 0.25 nmol/mg), but was still greatly reduced in comparison to wildtype (6.97 nmol/mg). The PtdIns, InsP(5) and InsP(6) levels were normal without treatment. Mrps6 gene expression was unaffected in the E18.5 knockout fetuses. This enigmatic model is not associated with neonatal PtdIns deficiency and rescue of the phenotype may be accomplished without restoration of Ins. The biochemical mechanism that both uniformly leads to death and allows for Ins rescue remains unknown. In conclusion, in neonatal brain tissue, Mrps6 gene expression may not be contingent on function of its embedded Slc5a3 gene, while inositide deficiency may not be the mechanism of lethal apnea in null Slc5a3 mice.

Effects of hyperosmolarity on the Na+ -myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line.

Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral Na(+)-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter (Na(+)-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment.

SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine.

This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.

Establishing a definitive stoichiometry for the Na+/monocarboxylate cotransporter SMCT1.

Several different stoichiometries have been proposed for the Na(+)/monocarboxylate cotransporter SMCT1, including variable Na(+)/substrate stoichiometry. In this work, we have definitively established an invariant 2:1 cotransport stoichiometry for SMCT1. By using two independent means of assay, we first showed that SMCT1 exhibits a 2:1 stoichiometry for Na(+)/lactate cotransport. Radiolabel uptake experiments proved that, unlike lactate, propionic acid diffuses passively through oocyte membranes and, consequently, propionate is a poor candidate for stoichiometric determination by these methods. Although we previously determined SMCT1 stoichiometry by measuring reversal potentials, this technique produced erroneous values, because SMCT1 simultaneously mediates both an inwardly rectifying cotransport current and an outwardly rectifying anionic leak current; the leak current predominates in the range where reversal potentials are observed. We therefore employed a method that compared the effect of halving the external Na(+) concentration to the effect of halving the external substrate concentration on zero-current potentials. Both lactate and propionate were cotransported through SMCT1 using 2:1 stoichiometries. The leak current passing through the protein has a 1 osmolyte/charge stoichiometry. Identification of cotransporter stoichiometry is not always a trivial task and it can lead to a much better understanding of the transport activity mediated by the protein in question.

Expression and functionality of the Na+/myo-inositol cotransporter SMIT2 in rabbit kidney.

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a "compatible osmolyte". Currently, three MI cotransporters have been identified: two are Na(+)-dependent (SMIT1 and SMIT2) and one is H(+)-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d-chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.

Membrane topology of loop 13-14 of the Na+/glucose cotransporter (SGLT1): a SCAM and fluorescent labelling study.

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na+/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES(-) and MTSET(+) had no effect on either cotransport or Na+ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K(i)(Pz) and K(m)(alphaMG) for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K(i)(Pz) values than wtSGLT1 with minimal changes in K(m)((alpha)MG), the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.

Determination of transport stoichiometry for two cation-coupled myo-inositol cotransporters: SMIT2 and HMIT.

Three different mammalian myo-inositol cotransporters are currently known; two are Na+-coupled (SMIT1 and SMIT2) and one is proton-coupled (HMIT). Although their transport stoichiometries have not been directly determined, significant cooperativities in the Na+ activation of SMIT1 and SMIT2 suggest that more than one Na+ ion drives the transport of each myo-inositol. The two techniques used here to determine transport stoichiometry take advantage of the electrogenicity of both SMIT2 and HMIT expressed in Xenopus oocytes. The first method compares the measurement of charge transferred into voltage-clamped oocytes with the simultaneous uptake of radiolabelled substrate. The second approach uses high accuracy volume measurements to determine the transport-dependent osmolyte uptake and compares it to the amount of charge transported. This method was calibrated using a potassium channel (ROMK2) and was validated with the Na+/glucose cotransporter SGLT1, which has a known stoichiometry of 2 : 1. Volume measurements indicated a stoichiometric ratio of 1.78 +/- 0.27 ion per alpha-methyl-glucose (alphaMG) for SGLT1 whereas the radiotracer uptake method indicated 2.14 +/- 0.05. The two methods yielded a SMIT2 stoichiometry measurement of 1.75 +/- 0.30 and 1.82 +/- 0.10, both in agreement with a 2 Na+:1 myo-inositol stoichiometry. For HMIT, the flux ratio was 1.02 +/- 0.04 charge per myo-inositol, but the volumetric method suggested 0.67 +/- 0.05 charge per myo-inositol molecule. This last value is presumed to be an underestimate of the true stoichiometry of one proton for one myo-inositol molecule due to some proton exchange for osmotically active species. This hypothesis was confirmed by using SGLT1 as a proton-driven glucose cotransporter. In conclusion, despite the inherent difficulty in estimating the osmotic effect of a proton influx, the volumetric method was found valuable as it has the unique capacity of detecting unidentified transported substrates.

Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells.

Myo-inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na(+)-dependent transporters such as SMIT1. We have recently identified a second Na(+)-myo-inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin-Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo-inositol by this cell line is 20-fold larger than that seen in native MDCK cells. The affinity for myo-inositol of MDCK cells transfected with SMIT2 is slightly lower (K(m)= 334 microm) than that found in voltage-clamped Xenopus laevis oocytes expressing SMIT2 (K(m)= 120 microm). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2-transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5-fold when submitted to 24 h hypertonic treatment (+200 mosmol l(-1)). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.

The human tumour suppressor gene SLC5A8 expresses a Na+-monocarboxylate cotransporter.

The orphan cotransport protein expressed by the SLC5A8 gene has been shown to play a role in controlling the growth of colon cancers, and the silencing of this gene is a common and early event in human colon neoplasia. We expressed this protein in Xenopus laevis oocytes and have found that it transports small monocarboxylic acids. The electrogenic activity of the cotransporter, which we have named SMCT (sodium monocarboxylate transporter), was dependent on external Na(+) and was compatible with a 3 : 1 stoichiometry between Na(+) and monocarboxylates. A portion of the SMCT-mediated current was also Cl(-) dependent, but Cl(-) was not cotransported. SMCT transports a variety of monocarboxylates (similar to unrelated monocarboxylate transport proteins) and most transported monocarboxylates demonstrated K(m) values near 100 microm, apart from acetate and d-lactate, for which the protein showed less affinity. SMCT was strongly inhibited by 1 mm probenecid or ibuprofen. In the absence of external substrate, a Na(+)-independent leak current was also observed to pass through SMCT. SMCT activity was strongly inhibited after prolonged exposure to high external concentrations of monocarboxylates. The transport of monocarboxylates in anionic form was confirmed by the observation of a concomitant alkalinization of the cytosol. SMCT, being expressed in colon and kidney, represents a novel means by which Na(+), short-chain fatty acids and other monocarboxylates are transported in these tissues. The significance of a Na(+)-monocarboxylate transporter to colon cancer presumably stems from the transport of butyrate, which is well known for having anti-proliferative and apoptosis-inducing activity in colon epithelial cells.

Identification of a novel Na+/myo-inositol cotransporter.

rkST1, an orphan cDNA of the SLC5 family (43% identical in sequence to the sodium myo-inositol cotransporter SMIT), was expressed in Xenopus laevis oocytes that were subsequently voltage-clamped and exposed to likely substrates. Whereas superfusion with glucose and other sugars produced a small inward current, the largest current was observed with myo-inositol. The expressed protein, which we have named SMIT2, cotransports myo-inositol with a K(m) of 120 microm and displays a current-voltage relationship similar to that seen with SMIT (now called SMIT1). The transport is Na(+)-dependent, with a K(m) of 13 mm. SMIT2 exhibits phlorizin-inhibitable presteady-state currents and substrate-independent "Na(+) leak" currents similar to those of related cotransporters. The steady-state cotransport current is also phlorizin-inhibitable with a K(i) of 76 microm. SMIT2 exhibits stereospecific cotransport of both d-glucose and d-xylose but does not transport fucose. In addition, SMIT2 (but not SMIT1) transports d-chiro-inositol. Based on previous publications, the tissue distribution of SMIT2 is different from that of SMIT1, and the existence of this second cotransporter may explain much of the heterogeneity that has been reported for inositol transport.