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.     

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues.

An indicator dilution technique with 22Na+ as the intravascular marker was used to measure unidirectional transport of D-[6-3H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, alpha-D-glucose has higher affinity for the carrier than does beta-D-glucose. A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Identification of the D-glucose binding polypeptide of the renal Na+-D-glucose cotransporter with a covalently binding D-glucose analog

The covalently binding D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG) was synthesised and shown to be a high-affinity inhibitor of the renal Na+-D-glucose contransporter. From renal brush-border membranes a protein fraction was isolated, in which the concentration of Na+-dependent phlorizin binding sites per mg protein was enriched 7-fold. In labeling experiments with this protein fraction a polypeptide of Mr approximately 79000 was identified as containing the D-glucose binding site of the renal Na+-D-glucose cotransporter.     

Distribution of sulfhydryl groups in intestinal brush border membranes. Localization of side-chains essential for glucose transport and phlorizin binding

1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4'-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5'-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4'-dithiodipyridine react with 5,5'-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 microM of the penetrating organomercurial p-chloromercuribenzoate, but was about ten times less sensitive to the poorly permeating p-chloromercuriphenylsulfonate. In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol L-glutathione (reduced form) could only revert the inhibition in leaky membranes.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

The glucose transport system of muscle plasma membranes: characterization by means of [3H]cytochalasin B binding

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092-1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific D-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [3H]CB binding activity (Kd of 0.28 microM). [3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by D-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [3H]CB binding. Higher concentrations of trypsin abolished [3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 mM) and N-ethylmaleimide (15 mM) inhibited [3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the D-glucose-sensitive [3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of Mr about 45K of muscle membranes which might represent the glucose carrier.     

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 2. Stabilization of an active conformation

Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na+/glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na+ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na+ and glucose were omitted. Na+ could not be replaced by K+, Rb+, or Li+. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na+ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na+-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na+-induced active conformer of the Na+/glucose symport system.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

High-affinity phlorizin binding to brush border membranes from small intestine: Identity with (a part of) the glucose transport system,dependence on the Na+-gradient,partial purification

In the presence of an NaSCN gradient phlorizin binds with a high affinity (K_d ? 4.7 μM) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for K_d corresponds closely to that of K_i determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min). The high affinity sites are related to glucose transport based on the similarity of the K_d and K_i values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2–3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein.     

Inactivation of the renal outer cortical brush-border membrane D-glucose transporter by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

The inactivation of the renal outer cortical brush-border membrane D-glucose transporter by the covalent carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is studied by monitoring its effects on sodium-dependent phlorizin binding to the active site of the carrier. In the presence of EEDQ, this component of phlorizin binding decreases exponentially and irreversibly with time. The order of this inactivation reaction is very close to 1, indicating that EEDQ modifies the transporter at a single essential site. This site can be partially protected by glucose and by other substrates of the transporter and completely protected by phlorizin, a nontransported competitive inhibitor. By contrast, sodium, a co-transported activator, has no protective effect. The concentration dependence of the protection provided by glucose and phlorizin indicates that the site of action of EEDQ is at or closely related to the substrate binding site on the carrier. The effects of EEDQ on the transporter are mimicked by another carboxyl specific reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The rate of inactivation of the transporter by EEDQ increases dramatically with decreasing pH, consistent with the hypothesis that the rate-limiting step in the inactivation process is a reaction with an essential carboxyl group. The properties of this group indicate, however, that it is distinct from the carboxyl group proposed by others as forming (a part of) the sodium binding site of sodium-coupled sugar carriers.     

Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.     

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of D-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 microM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 microM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexose transport by PhAsO. The data presented suggest a direct interaction between the D-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues

An indicator dilution technique with ²²Na⁺ as the intravascular marker was used to measure unidirectional transport of d-[6-³H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, α-d-glucose has higher affinity for the carrier than does β-d-glucose.A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Characteristics of Sorbitol Uptake in Rat Glial Primary Cultures

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.     

Cyclosporin binding to a protein component of the renal Na(+)-D-glucose cotransporter

The immunosuppressive and nephrotoxic agent cyclosporin binds to a renal polypeptide with an apparent molecular weight of 75,000 which has been identified as a component of the renal Na(+)-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H. (1987) J. Biol. Chem. 262, 10718-10729). The same Mr 75,000 polypeptide was covalently labeled with the D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside and with the cyclosporin analog N epsilon-(diazotrifluoroethyl)benzyl-D-Lys8- cyclosporin (CSDZ). CSDZ labeling was decreased when the brush-border membrane proteins were incubated with monoclonal antibodies against the Na(+)-D-glucose cotransporter. In the presence of 145 mM Na+, CSDZ labeling was decreased by D-glucose (1 microM, 1 mM, or 100 mM) and by phlorizin (100 or 500 microM). In the absence of Na+, CSDZ labeling was distinctly increased by 50 microM phlorizin and was slightly increased by 1 mM D-glucose, whereas CSDZ labeling was decreased by 50 microM phloretin and by 500 microM phlorizin. Furthermore, Na(+)-dependent high affinity phlorizin binding to the Na(+)-D-glucose cotransporter was competitively inhibited by cyclosporin A (Ki = 0.04 microM) while Na(+)-D-glucose cotransport was not influenced. The data suggest that a part of the cyclosporin binding domain on the Na(+)-D-glucose cotransporter is identical to the phloretin binding domain of the high affinity phlorizin binding site. While phloretin or the phloretin moiety of phlorizin may directly displace cyclosporin, interaction of D-glucose or of the D-glucose moiety of phlorizin with the transporter may alter the conformation of the cyclosporin binding site and this conformational change may be modulated by Na+.     

Bridging the gap between structure and kinetics of human SGLT1

The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.