A Ligand-dependent Conformational Change of the Na+/Galactose Cotransporter of Vibrio parahaemolyticus,Monitored by Tryptophan Fluorescence

Purification and reconstitution of the active Vibrio parahaemolyticus Na+/galactose transporter (vSGLT) enables us to do protein chemistry studies on a representative member of this class of membrane transporters. By measuring intrinsic tryptophan (Trp) fluorescence, conformational changes on the binding of substrates could be investigated. Trp fluorescence increased by 6% on the addition of saturating levels of both Na+ and D-galactose, with a K0.5 for D-galactose of 0.6 mM. No change was seen on the addition of Na+ alone or by adding D-galactose in the presence of K+. The Trp fluorescence could be quenched by acrylamide, but not by Cs+or I?. In the presence of Na+ or K+ alone, of Na+ or K+ and D-galactose, of Na+ and L-glucose, or in the absence of ligands, the fluorescence quenches by acrylamide were similar. This indicated that the tryptophan exposure to acrylamide was unchanged in the presence or absence of ligands. No shifts in lem maximum were observed. To find the Trp responsible for the change in fluorescence, Trp 448 in transmembrane helix 11 in the putative sugar-binding pocket was mutated. It was found that W448F showed a similar change in Trp fluorescence upon the addition of D-galactose in the presence of Na+. We conclude that the Trp fluorescence properties of the purified and reconstituted Na+/galactose cotransporter are selectively changed by the transported substrates Na+ and D-galactose, but it is not the Trp (W448) in the sugar translocation pathway that is involved.     

Conformational dynamics of hSGLT1 during Na+/glucose cotransport

This study examines the conformations of the Na(+)/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na(+) buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants tau(med) (3-20 ms, medium) and tau(slow) (15-70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (DeltaF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (DeltaF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zdelta (apparent valence of voltage sensor) approximately 1; and V(0.5) (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na(+)/glucose cotransport), and reduced maximal charge (Q(max)) and fluorescence (DeltaF(max)) with half-maximal concentrations (K(0.5)) of 1 mM. Increasing [alphaMDG](o) also shifted the V(0.5) for Q and DeltaF to more positive values, with K(0.5)'s approximately 1 mM. The major difference between Q and DeltaF was that at saturating [alphaMDG](o), the presteady-state current (and Q(max)) was totally abolished, whereas DeltaF(max) was only reduced 50%. Phlorizin reduced both Q(max) and DeltaF(max) (K(i) approximately 0.4 microM), with no changes in V(0.5)'s or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na(+)/sugar cotransport (saturating voltage and external Na(+) and sugar concentrations) is the slow release of Na(+) from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.     

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.     

Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter.

Conformational changes in the human Na(+)/glucose cotransporter (hSGLT1) were examined using hSGLT1 Q457C expressed in Xenopus laevis oocytes and tagged with tetramethylrhodamine-6-maleimide (TMR6M). Na(+)/glucose cotransport is abolished in the TMR6M-labeled mutant, but the protein binds Na(+) and sugar [Loo et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 7789-7794]. Under voltage clamp the fluorescence of labeled Q457C was dependent on external cations. Increasing [Na(+)] increased fluorescence with a Hill coefficient of 2 and half-maximal concentration (K(Na)(0.5)) of 49 mM at -90 mV. Li(+) also increased fluorescence, whereas choline, tetraethylammonium, and N-methyl-D-glucamine did not. Fluorescence was increased by sugars with specificity: methyl alpha-D-glucopyranoside > D-glucose > D-galactose > D-mannitol. Voltage-jump experiments (in 100 mM NaCl buffer in absence of sugar) elicited parallel changes in pre-steady-state charge movement and fluorescence. Charge vs voltage and fluorescence vs voltage curves followed Boltzmann relations with the same median voltage (V(0.5) = -50 mV), but the apparent valence was 1 for charge movement and 0.4 for fluorescence. V(0.5) for fluorescence and charge movement was shifted by -100 mV per 10-fold decrease in [Na(+)]. Under Na(+)-free conditions, there was a voltage-dependent change in fluorescence. Voltage-jump experiments showed that the maximal change in fluorescence increased 20% with sugar. These results indicate that Na(+), sugar, and membrane voltage change the local environment of the fluorophore at Q457C. Our interpretation of these results is (1) the conformational change of the empty transporter is voltage dependent, (2) two Na(+) ions can bind cooperatively to the protein before sugar, and (3) sugar binding induces a conformational change.     

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na(+), Li(+), and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.     

Voltage and substrate dependence of the inverse transport mode of the rabbit Na(+)/glucose cotransporter (SGLT1)

Properties of the cytoplasmic binding sites of the rabbit Na(+)/glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using alpha-methyl D-glucopyranoside (alphaMDG) as a substrate. The apparent affinity for alphaMDG depends on the cytoplasmic Na(+) concentration and voltage. At 0 mV the K(M) for alphaMDG is 7 mM at 110 mM Na(+) and 31 mM at 10 mM Na(+). The apparent affinity for alphaMDG and Na(+) is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half-maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.     

Sites important for Na+ and substrate binding in the Na+/proline transporter of Escherichia coli, a member of the Na+/solute symporter family.

To elucidate the functional importance of transmembrane domain II in the Na(+)/proline transporter (PutP) of Escherichia coli we analyzed the effect of replacing Ser-54 through Gly-58. Substitution of Asp-55 or Met-56 dramatically reduces the apparent affinity for Na(+) and Li(+) in a cation-dependent manner. Conversely, Cys in place of Gly-58 significantly reduces only the apparent proline affinity while substitution of Ser-57 results in a dramatic reduction of the apparent proline and cation affinities. Interestingly, upon increasing the proline concentration the apparent Na(+) affinity of Ser-57 replacement mutants converges toward the wild-type value, indicating a close cooperativity between cation and substrate site(s). This notion is supported by the fact that Na(+)-stimulated site-specific fluorescence labeling of a single Cys at position 57 is completely reversed by the addition of proline. Similar results are obtained upon labeling of a Cys at position 54 or 58. Taken together, these results indicate that Asp-55 and Met-56 are located at or close to the ion-binding site while Ser-54, Ser-57, and Gly-58 may be close to the proline translocation pathway. In addition, the data prod at an involvement of the latter residues in ligand-induced conformational dynamics that are crucial for cation-coupled transport.     

Sodium-dependent extracellular accessibility of Lys-84 in the sodium/dicarboxylate cotransporter

The Na(+)/dicarboxylate cotransporter transports Na(+) with citric acid cycle intermediates such as succinate and citrate. The present study focuses on transmembrane helix 3, which is highly conserved among the members of the SLC13 family. Fifteen amino acids in the extracellular half of transmembrane helix (amino acids 98-112) as well as Lys-84, previously shown to affect substrate affinity, were mutated individually to cysteine and expressed in the human retinal pigment epithelial cell line. Transport specificity ratio analysis shows that determinants for distinguishing succinate and citrate are found at amino acids Lys-84, Glu-101, Trp-103, His-106, and Leu-111. All of the mutants were tested for sensitivity to the membrane-impermeant cysteine-specific reagent (2-sulfonatoethyl) methanethiosulfonate (MTSES), but only K84C was sensitive to MTSES inhibition. The sensitivity of K84C to MTSES was greatest in the presence of sodium, and the inhibition could be prevented by addition of substrate or replacement of sodium, indicating that the accessibility of Lys-84 changes with conformational state. The substrate protection of MTSES inhibition of K84C appears to occur early in the transport cycle, before the large-scale conformational change associated with translocation of substrate. The results point to a new location for Lys-84 within the substrate access pore of the Na(+)/dicarboxylate cotransporter, either in a transmembrane helix or a reentrant loop facing a water-filled pore.     

Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique. For Q170C, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 50%. Decreased [Na(+)] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, tau, identical to wild-type. MTSES decreases maximal alpha-methyl D-glucopyranoside-induced currents by approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 60-70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the tau-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na(+) interaction. We conclude that 1), glutamine 170 lies in the Na(+) pathway in rabbit Na(+)/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.     

Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.     

Quantitative analysis of the interaction between the fluorescent probe eosin and the Na+/K+-ATPase studied through Rb+ occlusion

We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K(+)-congener Rb(+) in the Na(+)/K(+)-ATPase. Eosin decreases the equilibrium levels of occluded Rb(+), this effect being fully attributable to a decrease in the apparent affinity of the enzyme for Rb(+) since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na(+)/K(+)-ATPase, both to the Rb(+)-free and to the Rb(+)-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb(+) occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that, like ATP, it increases the velocity of deocclusion of Rb(+). Interactions between eosin and ATP on Rb(+)-release experiments seem to indicate that eosin binds to the low-affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.     

Partial deletion of a loop region in the high affinity K+ transporter HKT1 changes ionic permeability leading to increased salt tolerance

HKT1 is a high affinity K(+) transporter protein that is a member of a large superfamily of transporters found in plants, bacteria, and fungi. These transporters are primarily involved in K(+) uptake and are energized by Na(+) or H(+). HKT1 is energized by Na(+) but also mediates low affinity Na(+) uptake and may therefore be a pathway for Na(+) uptake, which is toxic to plants. The aim of this study was to identify regions of HKT1 that are involved in K(+)/Na(+) selectivity and alter the amino acid composition in those regions to increase the ionic selectivity of the transporter. A highly charged loop was identified, and two deletions were created that resulted in the removal of charged and uncharged amino acids. The functional changes caused by the deletions were studied in yeast and Xenopus oocytes. The deletions improved the K(+)/Na(+) selectivity of the transporter and increased the salt tolerance of the yeast cells in which they were expressed. In light of recent structural models of members of this symporter superfamily, it was necessary to determine the orientation of this highly charged loop. Introduction of an epitope tag allowed us to demonstrate that this loop faces the outside of the membrane where it is likely to facilitate the interaction with cations such as K(+) and Na(+). This study has identified an important structural feature in HKT1 that in part determines its K(+)/Na(+) selectivity. Understanding the structural basis of the functional characteristics in transporters such as HKT1 may have important implications for increasing the salt tolerance of higher plants.     

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters

The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.     

Examination of sodium/glucose cotransport by using a visible glucose analogue

The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes.

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.     

Structural Insights into Genetic Variants of Na+/Glucose Cotransporter SGLT1 Causing Glucose–Galactose Malabsorption: vSGLT as a Model Structure

Current advances in structural biology provide valuable insights into structure-function relationship of membrane transporters by solving crystal structures of bacterial homologs of human transporters. Therefore, scientists consider bacterial transporters as useful structural models for designing of drugs targeted in human diseases. The functional homology between Vibrio parahaemolyticus Na(+)/galactose transporter (vSGLT) and Na(+)/glucose cotransporter SGLT1 has been well established a decade ago. Now the crystal structure of vSGLT is considered quite valuable in explaining not only the cotransport mechanisms, but it also acts as a representative protein in understanding the protein stability and amino acid interactions within the core structure. We investigated the molecular mechanisms of genetic variations in SGLT1 that cause glucose-galactose malabsorption (GGM) defects using the crystal structure of vSGLT as a model sugar transporter. Our in silico mutagenesis and modeling analysis suggest that the GGM genetic variations lead to conformational changes either by structure destabilization or by formation of unnecessary interaction within the core structure of SGLT1 thereby explaining the genetic defects in Na(+) dependent sugar translocation across the cell membrane.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.     

Coupled sodium/glucose cotransport by SGLT1 requires a negative charge at position 454

Na(+)/glucose cotransport by SGLT1 is a tightly coupled process that is driven by the Na(+) electrochemical gradient across the plasma membrane. We have previously proposed that SGLT1 contains separate Na(+)- and glucose-binding domains, that A166 (in the Na(+) domain) is close to D454 (in the sugar domain), and that interactions between these residues influence sugar specificity and transport. We have now expressed the mutant D454C in Xenopus laevis oocytes and examined the role of charge on residue 454 by replacing the Asp with Cys or His, and by chemical mutation of D454C with alkylating reagents of different charge (MTSES(-), MTSET(+), MMTS(0), MTSHE(0), and iodoacetate(-)). Functional properties were examined by measuring sugar transport and cotransporter currents. In addition, D454C was labeled with fluorescent dyes and the fluorescence of the labeled transporter was recorded as a function of voltage and ligand concentration. The data shows that (1) aspartate 454 is critically important for the normal trafficking of the protein to the plasma membrane; (2) there were marked changes in the functional properties of D454C, i.e., a reduction in turnover number and a loss of voltage sensitivity, although there were no alterations in sugar selectivity or sugar and Na(+) affinity; (3) a negative charge on residue 454 increased Na(+) and sugar transport with a normal stoichiometry of 2 Na(+):1 sugar. A positive charge on residue 454, in contrast, uncoupled Na(+) and sugar transport, indicating the importance of the negative charge in the coordination of the cotransport mechanism.