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Ernest M Wright

Publications and source records attributed to Ernest M Wright.

At least 19 recordsLinked to original sources
PubMed2006

Imino sugars are potent agonists of the human glucose sensor SGLT3.

Imino sugars are used to treat type 2 diabetes mellitus [miglitol (Glyset)] and lysosomal storage disorders [miglustat (Zavesca)] based on the inhibition of alpha-glucosidases and glucosyltransferases. In this substrate specificity study, we examined the interactions of imino sugars with a novel human glucose sensor, sodium/glucose cotransporter type 3 (hSGLT3), using expression in Xenopus laevis oocytes and electrophysiology. The results for hSGLT3 are compared with those for alpha-glucosidases and human SGLT type 1 (hSGLT1), a well characterized sodium/glucose cotransporter of the SGLT family. In general, substrates have lower apparent affinities (K0.5) for hSGLT3 than hSGLT1 (D-glucose, alpha-methyl-D-glucose, 1-deoxy-D-glucose, and 4-deoxy-4-fluoro-D-glucose exhibit K0.5 values of 19, 21, 43, and 17 mM, respectively, for hSGLT3, and 0.5, 0.7, 10, and 0.07 mM, respectively, for hSGLT1). However, specificity of hSGLT3 binding is greater (D-galactose and 4-deoxy-4-fluoro-D-galactose are not hSGLT3 substrates, but have hSGLT1 K0.5 values of 0.6 and 1.3 mM). An important deviation from this trend is potent hSGLT3 activation by the imino sugars 1-deoxynojirimycin (DNJ), N-hydroxylethyl-1-deoxynojirimycin (miglitol), N-butyl-1-deoxynojirimycin (miglustat), N-ethyl-1-deoxynojirimycin, and 1-deoxynojirimycin-1-sulfonic acid, with K0.5 values of 0.5 to 9 microM. The diastereomer 1-deoxygalactonojirimycin activates hSGT3 with a K0.5 value of 11 mM, a 3000-fold less potent interaction than is observed for DNJ (4 microM). These imino sugar binding characteristics are similar to those for alpha-glucosidases, but there are no interactions with hSGLT1. This work provides insights into hSGLT3 and -1 substrate binding interactions, establishes a pharmacological profile to study endogenous hSGLT3, and may have important ramifications for the clinical application of imino sugars.

Animals↗
PubMed2006

Molecular interactions between dipeptides, drugs and the human intestinal H+ -oligopeptide cotransporter hPEPT1.

The human intestinal proton-coupled oligopeptide transporter hPEPT1 has been implicated in the absorption of pharmacologically active compounds. We have investigated the interactions between a comprehensive selection of drugs, and wild-type and variant hPEPT1s expressed in Xenopus oocytes, using radiotracer uptake and electrophysiological methods. The beta-lactam antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil, the antineoplastics delta-aminolevulinic acid (delta-ALA) and bestatin, and the neuropeptide N-acetyl-Asp-Glu (NAAG), were transported, as judged by their ability to evoke inward currents. When the drugs were added in the presence of the typical substrate glycylsarcosine (Gly-Sar), the inward currents were equal or less than that induced by Gly-Sar alone. This suggests that the drugs are transported at a lower turnover rate than Gly-Sar, but may also point towards complex interactions between dipeptides, drugs and the transporter. Gly-Sar and the drugs also modified the kinetics of hPEPT1 presteady-state charge movement, by causing a reduction in maximum charge (Qmax) and a shift of the midpoint voltage (V0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly-Sar > NAAG, delta-ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin. Based on steady-state and presteady-state analysis of Gly-Sar and cefadroxil transport, we proposed an extension of the 6-state kinetic model for hPEPT1 function that globally accounts for the observed presteady-state and steady-state kinetics of neutral dipeptide and drug transport. Our model suggests that, under saturating conditions, the rate-limiting step of the hPEPT1 transport cycle is the reorientation of the empty carrier within the membrane. Variations in rates of drug cotransport are predicted to be due to differences in affinity and turnover rate. Oral availability of drugs may be reduced in the presence of physiological concentrations of dietary dipeptides in the gut, suggesting that oral delivery drugs should be taken on an empty stomach. The common hPEPT1 single-nucleotide polymorphisms Ser117Asn and Gly419Ala retained the essential kinetic and drug recognition characteristics of the wild type, suggesting that neither variant is likely to have a major impact on oral absorption of drugs.

Animals↗
PubMed2006

A reinvestigation of the secondary structure of functionally active vSGLT, the vibrio sodium/galactose cotransporter.

The bacterial Na(+)/galactose cotransporter vSGLT of Vibrio parahaemolyticus is a member of the sodium:solute symporter family (SSS). Previous studies using electron microscopy have shown that vSGLT is a monomeric protein. Computational and experimental topological analyses have consistently indicated that this protein possesses 14 transmembrane alpha-helices. Our previous study using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to quantitate secondary structure content had indicated, in contrast, an alpha-helical content of only 35%, too little to be consistent with the 14-span model [le Coutre, J., et al. (2002) Biochemistry 41, 8082-6]. ATR-FTIR had also indicated that upon binding of Na(+) and d-galactose, the alpha-helical content increased to 53%. Here we revisit the vSGLT secondary structural distribution using an alternative approach, ultraviolet circular dichroism spectropolarimetry (CD), which is highly accurate in determining the alpha-helical content of a protein in solution. CD spectra were obtained from actively functional, soluble vSGLT and, as an internal check, from a fusion protein of vSGLT and the beta-barrel green fluorescent protein (GFP). Far-UV CD of vSGLT indicates a predominating 85% alpha-helical content, and an absence of beta-strands. Far-UV CD of the vSGLT-GFP fusion corroborates this profile, indicating an equivalent alpha-helical content, and a beta-strand content consistent with the GFP contribution. No detectable substrate-induced macroscopic changes in secondary structure are apparent in the far UV. In the near UV, increases in positive CD intensity occur in a stepwise manner with added substrates, implying changing environments of aromatic amino acid residues. CD thus confirms the current 14-transmembrane span model of vSGLT and reveals distinct substrate-induced conformational changes. The high percentage of alpha-helical structure found requires, when considered in the context of membrane topology, that nearly a third of the total alpha-helical fraction lies in extramembrane domains, which distinguishes this cotransporter from the unrelated lactose and glycerol 3-phosphate transporters.

Circular Dichroism↗
PubMed2006

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.

Animals↗
PubMed2004

Perturbation analysis of the voltage-sensitive conformational changes of the Na+/glucose cotransporter.

Conformational changes of the human Na(+)/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na(+)], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant tau = 0.17 ms) before decaying to steady state with two time constants (tau = 2-30 and 25-150 ms). The time to peak (0.9 ms) decreased with [Na(+)], and was not observed in 0 mM [Na(+)]. In absence of Na(+), charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na(+)/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.

Animals↗
PubMed2004

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.

Animals↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

Surprising versatility of Na+-glucose cotransporters: SLC5.

SLC5 is an ancient gene family with 11 members in the human genome. These membrane proteins have diverse, multiple functions ranging from actively transporting solutes, ions, and water, to channeling water and urea, to sensing glucose in cholinergic neurons. Metabolic disorders have been identified that are associated with congenital mutations in two of the human genes.

Animals↗
PubMed2003

A glucose sensor hiding in a family of transporters.

We have examined the expression and function of a previously undescribed human member (SGLT3/SLC5A4) of the sodium/glucose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na+-dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na+/glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.

Animals↗
PubMed2003

Functional asymmetry of the human Na+/glucose transporter (hSGLT1) in bacterial membrane vesicles.

The functional characteristics of the forward and reverse transport modes of the human Na(+)/glucose transporter (hSGLT1) were investigated using plasma membrane vesicles of E. coli expressing the recombinant transporter. Correctly and inverse-oriented vesicles were employed to measure the initial rates of methyl-alpha-D-glucose uptake, under zero-trans conditions, as a function of Na(+), sugar, and phlorizin concentrations and membrane potential. This approach enabled the analysis of the two faces of hSGLT1 in parallel, revealing the reversibility of Na(+)/sugar cotransport. While the key characteristics of secondary active sugar transport were maintained in both modes, namely, Na(+) and voltage dependence, the kinetic properties of the two sides indicated a functional asymmetry of the transporter. That is, the apparent affinity for sugar and driver cation Na(+) exhibited a difference of more than 1 order of magnitude between the two modes. Furthermore, the selectivity pattern of ligands and the interaction of the transporter with the competitive inhibitor phlorizin were different. Whereas the high-affinity substrates, D-glucose and D-galactose, inhibited uptake of radioactive sugar tracer at their physiological concentrations (10 mM) in the forward reaction, they were poor inhibitors even at high concentrations in the reverse transport mode. Taken together, these results confirm the successful employment of E. coli to express and characterize a human membrane protein (hSGLT1), elucidating the functional asymmetry of this cotransporter.

Cell Membrane↗
PubMed2003

The sodium/glucose cotransport family SLC5.

The sodium/glucose cotransporter family (SLCA5) has 220 or more members in animal and bacterial cells. There are 11 human genes expressed in tissues ranging from epithelia to the central nervous system. The functions of nine have been revealed by studies using heterologous expression systems: six are tightly coupled plasma membrane Na(+)/substrate cotransporters for solutes such as glucose, myo-inositol and iodide; one is a Na(+)/Cl(-)/choline cotransporter; one is an anion transporter; and another is a glucose-activated ion channel. The exon organization of eight genes is similar in that each comprises 14-15 exons. The choline transporter (CHT) is encoded in eight exons and the Na(+)-dependent myo-inositol transporter (SMIT) in one exon. Mutations in three genes produce genetic diseases (glucose-galactose malabsorption, renal glycosuria and hypothyroidism). Members of this family are multifunctional membrane proteins in that they also behave as uniporters, urea and water channels, and urea and water cotransporters. Consequently it is a challenge to determine the role(s) of these genes in human physiology and pathology.

Amino Acid Sequence↗
PubMed2003

Structure of functional single AQP0 channels in phospholipid membranes.

Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.

Animals↗
PubMed2003

Intestinal absorption in health and disease--sugars.

Carbohydrates are mostly digested to glucose, fructose and galactose before absorption by the small intestine. Absorption across the brush border and basolateral membranes of enterocytes is mediated by sodium-dependent and -independent membrane proteins. Glucose and galactose transport across the brush border occurs by a Na(+)/glucose (galactose) co-transporter (SGLT1), whereas passive fructose transport is mediated by a uniporter (GLUT5). The passive exit of all three sugars out of the cell across the basolateral membrane occurs through two uniporters (GLUT2 and GLUT5). Mutations in SGLT1 cause a major defect in glucose and galactose absorption (glucose-galactose Malabsorption), but mutations in GLUT2 do not appear to disrupt glucose and galactose absorption. Studies on GLUT1 null mice and Fanconi-Bickel patients suggest that there is another exit pathway for glucose and galactose that may involve exocytosis. There are no known defects of fructose absorption.

Animals↗
PubMed2002

Water pumps.

The transport of water across epithelia has remained an enigma ever since it was discovered over 100 years ago that water was transported across the isolated small intestine in the absence of osmotic and hydrostatic pressure gradients. While it is accepted that water transport is linked to solute transport, the actual mechanisms are not well understood. Current dogma holds that active ion transport sets up local osmotic gradients in the spaces between epithelial cells, the lateral intercellular spaces, and this in turn drives water transport by local osmosis. In the case of the small intestine, which in humans absorbs about 8 l of water a day, there is no direct evidence for either local osmosis or aquaporin gene expression in enterocytes. Intestinal water absorption is greatly enhanced by glucose, and this is the basis for oral rehydration therapy in patients with secretory diarrhoea. In our studies of the intestinal brush border Na+-glucose cotransporter we have obtained evidence that there is a direct link between the transport of Na+, glucose and water transport, i.e. there is cotransport of water along with Na+ and sugar, that will account for about 50 % of the total water transport across the human intestinal brush border membrane. In this short review we summarize the evidence for water cotransport and propose how this occurs during the enzymatic turnover of the transporter. This is a general property of cotransporters and so we expect that this may have wider implications in the transport of water and other small polar molecules across cell membranes in animals and plants.

Animals↗
PubMed2002

Ligand-induced differences in secondary structure of the Vibrio parahaemolyticus Na+/galactose cotransporter.

A detailed structural study of the prokaryotic sodium/galactose transporter (vSGLT) from Vibrio parahaemolyticus using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy reveals stepwise increases in alpha-helicity upon binding of sodium and D-galactose. These increases in helicity correlate with decreases in beta-structural elements. The changes are accompanied by stepwise reductions in the degree of H/D exchange (HDX), suggesting reduced accessibility of water to the protein backbone. The data demonstrate discrete conformational changes from one intermediate to the next during the catalytic cycle of the protein and are interpreted in a model of the symport reaction mechanism.

Amides↗
PubMed2002

Employing Escherichia coli to functionally express, purify, and characterize a human transporter.

Large-scale purification of recombinant human membrane proteins represents a rate-limiting step toward the understanding of their role in health and disease. There are only four mammalian membrane proteins of known structure, and these were isolated from natural sources (see http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html). In addition, genetic diseases of membrane proteins are frequently caused by trafficking defects, and it is enigmatic whether these mutants are functional. Here, we report the employment of Escherichia coli for the functional expression, purification, and reconstitution of a human membrane protein, the human Na+/glucose cotransporter (hSGLT1). The use of an E. coli mutant defective in the outer membrane protease OmpT, incubation temperatures below 20 degrees C, and transcriptional regulation from the lac promoter/operator are crucial to reduce proteolytic degradation. Purification of a recombinant hSGLT1 through affinity chromatography yields about 1 mg of purified recombinant hSGLT1 per 3 liters of cultured bacterial cells. Kinetic analysis of hSGLT1 in proteoliposomes reveals that a purified recombinant transporter, which is missorted in eukaryotic cells, retains full catalytic activity. These results indicate the power of bacteria to manufacture and isolate human membrane proteins implicated in genetic diseases.

Cloning, Molecular↗
PubMed2002

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.

Animals↗