Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

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.     

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.     

Isolation and reconstitution of the intestinal Na+/glucose cotransporter

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.     

Evidence for tyrosyl residues at the Na+ site on the intestinal Na+/glucose cotransporter

A tyrosine group has been identified at, or near, the Na+-binding site of the Na+/glucose and Na+/proline cotransporters of rabbit intestinal brush-borders. Three tyrosine group-specific reagents, n-acetylimidazole, tetranitromethane, and p-nitrobenzene sulfonyl fluoride, were used to evaluate the role of tyrosyl groups in Na+-dependent glucose transport, Na+-dependent phlorizin binding, and the Na+-induced fluorescence quenching of fluorescein isothiocyanate bound to the glucose site of the carrier. All three reagents inhibited glucose transport, phlorizin binding, and fluorescein isothiocyanate quenching by 50-85% with Ki values in the range 7-50 microM. The presence of Na+ during the exposure of membranes to the reagents completely protected against inhibition, the Na+ concentration required to produce 50% protection was 14-36 mM. Fluorescent derivatives of n-acetylimidazole were synthesized to identify the tyrosyl residues on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A total of five polypeptide bands were labeled with eosin or fluorescein n-acetylimidazole in a Na+-sensitive manner. Two of these bands, previously identified as the glucose (75,000-dalton) and proline (100,000-dalton) binding sites of the glucose and proline carriers, account for 50% of the Na+-sensitive tyrosyl residues. On the basis of these studies, we believe that the Na+/glucose cotransporter contains both the Na+ and glucose active sites on the same polypeptide or that the cotransporter consists of two similar polypeptides, each containing one substrate binding site.     

Local conformational changes in the Vibrio Na+/galactose cotransporter

Na(+) and sugar transport by cotransporters (symporters) is thought to occur as a series of ordered ligand-induced conformational changes. To localize these conformational changes in a bacterial Na(+)/galactose cotransporter, we have employed a combination of cysteine-scanning and fluorescence techniques. Single or pairs of cysteine residues were introduced into the external face of a cysteine-less Vibrio parahaemolyticus sodium/glucose cotransporter for expression in Escherichia coli, and each transporter was purified using affinity chromatography. All the mutant proteins retained transport activity in bacteria and proteoliposomes. Each mutant was exposed to two different fluorescence reagents, ThioGlo3 or pyrene maleimide, that are essentially nonfluorescent until they react with a thiol. Fluorescence was recorded as a function of time and ligand concentrations. The reagents specifically labeled six of the seven cysteine mutants, but only in Cysteine 423 was the fluorescence affected by ligands. The rate of labeling of Cys423 by ThioGlo3 or pyrene maleimide was reduced by D-galactose in Na(+) buffer. Furthermore, the fluorescence of Thioglo3-labeled Cys423 was quenched by D-galactose, but only in the presence of Na(+). This quench was not accompanied by a Stokes shift and was not produced by nontransported sugars, e.g., L-glucose. Reducing the sodium concentration from 200 to 10 mM decreased the apparent affinity for d-galactose without altering the maximum quench with saturating D-galactose. Reducing the galactose concentration from 20 to 0.5 mM reduced both the apparent affinity for Na(+) and the maximum quench at saturating Na(+). These results suggest an ordered reaction scheme with Na(+) binding first. The fluorescence results with ThioGlo3-labeled Cys423 indicate that conformational changes underlying Na(+)/galactose cotransport occur at or near the extracellular domain between transmembrane helices 10 and 11.     

Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein.

Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.     

Investigating the conformational states of the rabbit Na+/glucose cotransporter

The Na(+) and voltage-dependence of transient rabbit Na(+)/glucose cotransporter (rSGLT1) kinetics was studied with the two-electrode voltage-clamp technique and Xenopus laevis oocytes. Using step changes in membrane potential, in the absence of glucose but with 100 or 10 mM Na(+), transient currents were measured corresponding to binding/debinding of Na(+) and conformational changes of the protein. Previously, only a single time constant has been published for rSGLT1. We, however, observed three decay components; a fast (tau(f), 0.5-1 ms) voltage- and Na(+)-independent decay, and medium (tau(m), 0.5-4 ms) and slow (tau(s), 8-50 ms) voltage- and Na(+)-dependent decays. Transient currents were simulated and fit using a four-state model to obtain kinetic parameters for the system. The four-state model was able to reconstitute an assortment of experimental data.     

Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO(3)(-)/cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO(3)(-) has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO(3)(-) but not Cl(-) induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO(3)(-)-dependent hyperpolarization was not observed when Na(+) was replaced by the non-permeant cation choline(+). Replacement of Na(+) by choline(+) also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO(3)(-) current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na(+) suggest that a Na(+)/HCO(3)(-) cotransporter is present in mouse sperm and is coupled to events regulating capacitation.     

On the stoichiometry of intercellular Na+/K(+)-exchange

Literature data on the atomic and ionic sizes of sodium (Na+) and potassium (K+), and the thermodynamic, and physicochemical characteristics of solutions containing Na+ and K+ have been compared. It was shown that in solutions containing Na+ only the activity of H+ is 1.31 +/- 0.038 times higher than in solutions containing only K+. It was concluded that there is reason to believe that the stoichiometry of Na+/K(+)-exchange is close to 3:4. The different degree of hydratation of Na+ and K+ caused by different density of their charges is considered to be the main reason for the result obtained.     

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

Summary The 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--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 (K _i 6 and 9 m), and saturable (-methyl-d-glucopyranosideK _m 110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-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.     

Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1)

We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/microg total RNA) relative to that in the kidney (3E + 4 molecules/microg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/microg total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/microg total RNA (in skeletal muscle) to 3.2E + 6 molecules/microg total RNA (in kidney), levels 10-100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart.     

Examination of substrate-induced conformational changes in the Na+/glucose cotransporter

Conformations of the Na+/glucose cotransporter were examined using tryptophan fluorescence and substrates to induce cotransporter conformational changes. Addition of Na+ but not K+ or TMA+ resulted in a saturable quenching of tryptophan fluorescence with a K0.5 for Na+ of 28 mM. In the presence of saturating Na+ concentrations, d-glucose but not l-glucose, fructose, or phlorizin resulted in a partial return of tryptophan fluorescence to approximately 70% of the substrate-free levels. This return of tryptophan fluorescence was a saturable function of d-glucose concentration with a K0.5 of 43 microM. The three conformations were compared with respect to their sensitivity to tryptophan quench reagents. Acrylamide quenching was unaffected by substrates. In contrast, I- quenching decreased 40% in the presence of Na+, while Cs+ quenching increased 64%. Addition of saturating d-glucose concentrations resulted in the return of I- quenching to 90% of the substrate-free values and reduced Cs+ quenching to substrate-free levels. In contrast, phlorizin did not mimic the effect of d-glucose on tryptophan fluorescence. These results are interpreted in terms of a second substrate-induced cotransporter conformational change which based on similar substrate specificities appears directly related to cotransporter-mediated Na+ and d-glucose transport.