Michael J. Coady

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 Km values near 100 μm, 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 aK m of 120 μm and displays a current-voltage relationship similar to that seen with SMIT (now called SMIT1). The transport is Na+-dependent, with aK 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 μm. SMIT2 exhibits stereospecific cotransport of bothd-glucose and d-xylose but does not transport fucose. In addition, SMIT2 (but not SMIT1) transportsd-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.

A Loss-of-Function Mutation in NaPi-IIa and Renal Fanconi's Syndrome

We describe two siblings from a consanguineous family with autosomal recessive Fanconis 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.

Functional expression of tagged human Na+—glucose cotransporter in Xenopus laevis oocytes

1 High‐affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure‐ function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2 Six constructs, made from three tags (myc, haemaglutinin and poly‐His) inserted at both the C‐ and N‐terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N‐terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc‐tagged and the untagged hSGLT1 proteins. 3 On the basis of both steady‐state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady‐state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4 The myc‐tagged hSGLT1 was also successfully expressed in polarized MDCK cells. α‐Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5 These comparative studies demonstrate that the myc epitope inserted at the N‐terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.

Electrogenic amino acid exchange via the rBAT transporter

A cDNA clone was isolated from rabbit renal cortex using DNA‐mediated expression cloning, which caused alanine‐dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na‐independent amino acid transporter previously cloned elsewhere. Exposure of cDNA‐injected oocytes to neutral amino acids led to voltage‐dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut‐open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter

The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.

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 α‐methyl‐glucose (αMG) 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.

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.

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 (Km= 334 μm) than that found in voltage‐clamped Xenopus laevis oocytes expressing SMIT2 (Km= 120 μm). 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.