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.

The transport mechanism of the human sodium/myo-inositol transporter 2 (SMIT2/SGLT6), a member of the LeuT structural family.

The sodium/myo-inositol transporter 2 (SMIT2) is a member of the SLC5A gene family, which is believed to share the five-transmembrane segment inverted repeat of the LeuT structural family. The two-electrode voltage-clamp (TEVC) technique was used to measure the steady-state and the pre-steady-state currents mediated by human SMIT2 after expression in Xenopus laevis oocytes. Phlorizin is first shown to be a poor inhibitor of pre-steady-state currents for depolarizing voltage pulse. From an up to threefold difference between the apparent ON and OFF transferred charges during a voltage pulse, we also show that a fraction of the transient current recorded for very negative potentials is not a true pre-steady-state current coming from the cotransporter conformational changes. We suggest that this transient current comes from a time-dependent leak current that can reach large amplitudes when external Na(+) concentration is reduced. A kinetic model was generated through a simulated annealing algorithm. This algorithm was used to identify the optimal connectivity among 19 different kinetic models and obtain the numerical values of the associated parameters. The proposed 5-state model includes cooperative binding of Na(+) ions, strong apparent asymmetry of the energy barriers, a rate-limiting step that is likely associated with the translocation of the empty transporter, and a turnover rate of 21 s(-1). The proposed model is a proof of concept for a novel approach to kinetic modeling of electrogenic transporters and allows insight into the transport mechanism of members of the LeuT structural family at the millisecond timescale.

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.

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.

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique.

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.

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.

Ammonium transport and pH regulation by K(+)-Cl(-) cotransporters.

The Na(+)-K(+)-Cl(-) cotransporters (NKCCs), which belong to the cation-Cl(-) cotransporter (CCC) family, are able to translocate NH4(+) across cell membranes. In this study, we have used the oocyte expression system to determine whether the K(+)-Cl(-) cotransporters (KCCs) can also transport NH4(+) and whether they play a role in pH regulation. Our results demonstrate that all of the CCCs examined (NKCC1, NKCC2, KCC1, KCC3, and KCC4) can promote NH4(+) translocation, presumably through binding of the ion at the K(+) site. Moreover, kinetic studies for both NKCCs and KCCs suggest that NH4(+) is an excellent surrogate of Rb(+) or K(+) and that NH4(+) transport and cellular acidification resulting from CCC activity are relevant physiologically. In this study, we have also found that CCCs are strongly and differentially affected by changes in intracellular pH (independently of intracellular [NH4(+)]). Indeed, NKCC2, KCC1, KCC2, and KCC3 are inhibited at intracellular pH <7.5, whereas KCC4 is activated. These results indicate that certain CCC isoforms may be specialized to operate in acidic environments. CCC-mediated NH4(+) transport could bear great physiological implication given the ubiquitous distribution of these carriers.

Glucose accumulation can account for the initial water flux triggered by Na+/glucose cotransport.

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na+ influx failed to reproduce the large and rapid cell swelling induced by Na+/solute cotransport. We have investigated this phenomenon by comparing a Na+/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K+ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na+/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na+/glucose cotransport activation without having to propose the presence of secondary active water transport.

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.

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.