Experimental comparison between tractional and compressive stress on temporomandibular joint.

OBJECTIVE: We experimentally compared the effects of compressive and tractional mechanical stress on the temporomandibular joint (TMJ) of rabbits to assess the etiology of progressive condylar resorption. MATERIALS AND METHODS: We performed a cortical osteotomy using custom-made devices that were lengthened by 0.25 mm every 12 h for 1 week after surgery. During this time, the rabbit TMJ was under compressive or tractional mechanical stress. The samples in each group were examined using micro-computed tomography and histological staining. RESULTS: Scores for the area of bone resorption were higher in the compressive group than in the tractional group. Moreover, scores for the depth of bone resorption were higher in the compressive group than those in the tractional group. We observed a significantly higher prevalence of resorption in the subcondylar bone in the compressive group than in the tractional group. There were substantially more cells that were positive for tartrate-resistant acid phosphatase in the compressive group than in the control and tractional groups. CONCLUSIONS: The outcomes here suggest that excessive mechanical stress, particularly compressive mechanical stress, may significantly affect morphological bone change findings in the TMJ.

Active sugar transport in health and disease.

Secondary active glucose transport occurs by at least four members of the SLC5 gene family. This review considers the structure and function of two premier members, SGLT1 and SGLT2, and their role in intestinal glucose absorption and renal glucose reabsorption. Genetics disorders of SGLTs include Glucose-Galactose Malabsorption, and Familial Renal Glucosuria. SGLT1 plays a central role in Oral Rehydration Therapy used so effectively to treat secretory diarrhoea such as cholera. Increasing attention is being focused on SGLTs as drug targets for the therapy of diabetes.

Residue 457 controls sugar binding and transport in the Na(+)/glucose cotransporter.

The Na(+)/glucose cotransporter (SGLT1) is highly selective for its natural substrates, d-glucose and d-galactose. We have investigated the structural basis of this sugar selectivity on the human isoform of SGLT1, single site mutants of hSGLT1, and the pig SGLT3 isoform, expressed in Xenopus oocytes using electrophysiological methods and the effects of cysteine-specific reagents. Kinetics of transport of glucose analogues, each modified at one position of the pyranose ring, were determined for each transporter. Correlation of kinetics with amino acid sequences indicates that residue Gln-457 sequentially interacts with O1 of the pyranose in the binding site, and with O5 in the translocation pathway. Furthermore, correlation of the selectivity characteristics of the SGLT isoforms (SGLT1 transports both glucose and galactose, but SGLT2 and SGLT3 transport only glucose) with amino acid sequence differences, suggests that residue 460 (threonine in SGLT1, and serine in SGLT2 and SGLT3) are involved in hydrogen bonding to O4 of the pyranose. In addition, the results show that substrate specificity of binding is not correlated to substrate specificity of transport, suggesting there are at least two steps in the sugar translocation process.

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl-/GABA (hGAT1) cotransporters.

1. Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na(+)/glucose (hSGLT1) and Na(+)/Cl(-)/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. 2. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. 3. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7x12A. Important hydrogen bond interactions occur at five positions bordering this surface. 4. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site approximately 8A from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). 5. Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors.

Evidence for the involvement of Ala 166 in coupling Na(+) to sugar transport through the human Na(+)/glucose cotransporter.

We mutated residue 166, located in the putative Na(+) transport pathway between transmembrane segments 4 and 5 of human Na(+)/glucose cotransporter (hSGLT1), from alanine to cysteine (A166C). A166C was expressed in Xenopus laevis oocytes, and electrophysiological methods were used to assay function. The affinity for Na(+) was unchanged compared to that of hSGLT1, whereas the sugar affinity was reduced and sugar specificity was altered. There was a reduction in the turnover rate of the transporter, and in contrast to that of hSGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, indicating that the residue is always accessible from the extracellular surface. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTSET, and MTSES all changed the pre-steady-state kinetics of A166C, independent of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na(+) and sugar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na(+) and sugar transport pathways.

Human cerebral cysticercosis: immunolocalization of a sodium-dependent glucose cotransporter (SGLT) in larval and adult tapeworms.

Light microscopic immunocytochemistry was used to examine human brain cysticerci resected from the fourth ventricles of patients who had not been treated with anthelminthic drugs. Tissues were examined from 3 different patients undergoing surgery for treatment of hydrocephalus. A rabbit polyclonal antiserum to the peptide corresponding to amino acids 564-575 unique to the rabbit sodium-dependent, SGLT1 glucose cotransporter labeled with immunoperoxidase, localized immunoreactive SGLT epitopes. This antibody localizes SGLT1 in the apical brush borders of human enterocytes, but is negative in cytoplasm, as well as lateral and basal enterocyte membranes. Taenia solium neurocysticerci were SGLT positive; transporter protein was highly expressed on the surface microvilli of the external cyst wall. The well-developed network of small and larger osmoregulatory ducts within racemose larval cystcerci displayed high expression of SGLT cotransporter, consistent with a resorptive function for this system of tubules. Because water is cotransported with glucose molecules by the SGLT protein, its high expression in neurocysticerci may contribute to the expansive growth of these larvae in subarachnoid and intraventricular sites. The SGLT epitopes were also immunolocalized in gravid proglottids of Taenia saginata, indicating that cotransporter expression persisted in intestinal-dwelling, adult tapeworms. Cotransporter antibody was abundantly localized at the proglottid tegumentary surface and in the lateral osmoregulatory ducts, analogous to the SGLT localization in cysticerci. Furthermore, high expression of this cotransporter was seen in the branches of the uterus, suggesting that SGLT-mediated absorption of glucose and water has an important functional role within the reproductive system of adult tapeworms.

Na+-to-sugar stoichiometry of SGLT3.

Sodium-glucose cotransporters (SGLTs) mediate active transport of sugar across cell membranes coupled to Na+, by using the electrochemical gradient as a driving force. In the kidney, there is evidence for two kinds of cotransporters, a high-affinity, low-capacity system, and a low-affinity, high-capacity system, with differences in substrate specificity and kinetics. Three renal SGLT clones have been identified: SGLT1 corresponding to the high-affinity system, and SGLT2 and SGLT3 with properties reminiscent of the low-affinity system. We have determined the stoichiometry of pig SGLT3 (pSGLT3) by using a direct method, comparing the substrate-induced inward charge to 22Na or [14C]alpha-methyl-D-glucopyranoside uptake in the same oocyte. pSGLT3 stoichiometry is 2 Na+:1 sugar, the same as that for SGLT1, but different from SGLT2 (1:1). The Na+ Hill coefficient for SGLT3 is approximately 1.5, suggesting low cooperativity between Na+ binding sites. Thus SGLT3 has functional characteristics intermediate between SGLT1 and SGLT2, so, whereas SGLT3 stoichiometry is the same as that for SGLT1 (2:1), sugar affinity and specificity are similar to SGLT2.

Urea transport by cotransporters.

The rabbit Na+-glucose cotransporter (rbSGLT1) was expressed in Xenopus laevis oocytes and urea transport in rbSGLT1 and non-injected (control) oocytes was studied using [14C]urea as a tracer. The level of rbSGLT1 expression in these batches of oocytes was monitored by measuring the uptake of alpha-methyl-D-[14C]glucopyranoside ([14C]alphaMDG). In rbSGLT1-expressing oocytes, there was a 4-fold increase in urea transport in the absence of sugar relative to that in control oocytes. Urea uptake was not Na+ dependent and was linear with both time of incubation (5-120 min) and increasing urea concentration (50 microM to 100 mM) in the bathing medium. rbSGLT1 urea uptake was blocked by the rbSGLT1-specific inhibitor phlorizin (Ki 1 microM) in 100 mM NaCl buffer, but was not affected in 100 mM choline chloride buffer. Phloretin inhibited rbSGLT1 urea uptake with a low affinity (Ki > 1 mM) in the presence and absence of Na+. The uptake of 55 m[mu]M urea through rbSGLT1 was not blocked by 100 mM urea analogues including thiourea, 1,3-dimethyl urea, 1,1-dimethyl urea and acetamide. The activation energies (Ea) of urea transport for control and rbSGLT1-expressing oocytes were 14+/-3 and 6+/-1 kcal mol(-1), respectively. The low Ea for urea transport through rbSGLT1 is comparable to the Ea of passive water transport through rbSGLT1. Urea transport through rbSGLT1 was further increased when the cotransporter was activated by the addition of sugar to the external medium. The rate of sugar-dependent urea uptake was directly proportional to the rate of Na+-glucose-H2O cotransport such that the amount of urea transport was approximately proportional to the molar concentration ratio of urea to H2O (55 microM/55 M). The low affinity Na+-glucose (pSGLT3), the Na+-iodide (rNIS) and the Na+-(Cl-)-GABA (hGAT1) cotransporters expressed in oocytes demonstrated similar urea transport properties. These observations suggest that cotransporters behave as urea channels in the absence of substrates. Furthermore, under substrate-transporting conditions, the same cotransporters serve as urea cotransporters. This could account for urea transport in cells that appear not to have urea uniporters or channels.

Glycoside binding and translocation in Na(+)-dependent glucose cotransporters: comparison of SGLT1 and SGLT3.

Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na(+)/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K(0.5) 0.06 mm) and 2-naphthylglucose with lower affinity (K(0.5) 0. 5 mm) than alpha-methyl-d-glucopyranoside (alpha MDG, 0.2 mm). Both were poorly transported (maximal velocities, I(max), 14% and 8% of alpha MDG). Other compounds were inhibitors (K(i)s 1-13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than alpha MDG (K(0.5)s 0.9, 0.2 and 2.5 mm and relative I(max)s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I(max)s (130 and 120%) and comparable K(0. 5)s (8 and 1 mm). Increased affinity of indican relative to alphaMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value.

Purification and functional reconstitution of a truncated human Na(+)/glucose cotransporter (SGLT1) expressed in E. coli.

A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture. Reconstitution of both GST-C(5) and C(5) proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [(3)H]D-glucose into C(5)-proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C(5) in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.

Passive water and ion transport by cotransporters.

1. The rabbit Na+-glucose (SGLT1) and the human Na+-Cl--GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker-sensitive Na+ currents in the absence of substrates (glucose and GABA). 2. Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3-5 microM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 microM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 3. The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol-1, respectively. The high Ea for Na+ transport is comparable to that of Na+-glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 4. GAT1 also behaved as an SKF89976A-sensitive water channel. We did not observe passive Na+ transport through GAT1. 5. We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.

Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects.

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.

Structure and function of the Na+/glucose cotransporter.

Cotransporters are a major class of membrane transport proteins that are responsible for the accumulation of nutrients, neurotransmitters, osmolytes and ions in cells from bacteria to man. The energy for solute accumulation comes from the proton and/or sodium electrochemical gradients that exist across cell membranes. A major problem in biology is how transport is coupled to these electrochemical potential gradients. The primary example of this class of membrane proteins is the intestinal brush border Na+/glucose cotransporter (SGLT1), first described by Bob Crane in 1960. Over 35 members of the SGLT1 gene family have been identified in animal cells, yeast and bacteria, and all share a common core structure of 13 transmembrane (TM) helices. Electrophysiological techniques have been used to examine the function of several family members, chimeras and mutants expressed in heterologous systems such as Xenopus laevis oocytes. These have revealed that cotransporters are multi-functional proteins: they are responsible for 1). uncoupled passive Na+ transport (Na+ uniport); 2). down-hill water transport in the absence of substrate; 3). Na+/substrate cotransport; and 4). Na+/substrate/water cotransport. The sugar binding and translocation pathway is formed by 4 TM helices near the C-terminal of the protein, helices 10-13. We propose that the N-terminal domains of SGLT1 are responsible for Na+ binding and/or translocation, and that Na+/glucose cotransport results from interactions between the N- and C-terminal domains of the protein.

Sodium and lithium interactions with the Na+/Dicarboxylate cotransporter.

The two-electrode voltage clamp was used to study the currents associated with transport of succinate by the cloned Na+/dicarboxylate cotransporter, NaDC-1, expressed in Xenopus oocytes. The presence of succinate induced inward currents which were dependent on the concentrations of succinate and sodium, and on the membrane potential. At -50 mV, the K0.5succinate was 180 microM and the K0.5Na+ was 19 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na+:1 divalent anion substrate. Currents were induced in NaDC-1 by a range of di- and tricarboxylates, including citrate, methylsuccinate, fumarate, and tricarballylate. Although Na+ is the preferred cation, Li+ was also able to support transport. The K0.5succinate was approximately 10-fold higher in Li+ compared with Na+. In the presence of Na+, however, Li+ was a potent inhibitor of transport. Millimolar concentrations of Li+ resulted in decreases in apparent succinate affinity and in the Imaxsuccinate. Furthermore, lithium inhibition under saturating sodium concentrations showed hyperbolic kinetics, suggesting that one of the three cation binding sites in NaDC-1 has a higher affinity for Li+ than Na+. We conclude that NaDC-1 is an electrogenic anion transporter that accepts either Na+ or Li+ as coupling cations. However, NaDC-1 contains a single high affinity binding site for Li+ that, when occupied, results in transport inhibition, which may account for its potent inhibitory effects on renal dicarboxylate transport.

Conformational changes couple Na+ and glucose transport.

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady-state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady-state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Na/D-glucose cotransport and SGLT1 expression in hen colon correlates with dietary Na+.

We have tested whether separately varying the content of either Na or Cl in diets causes earlier observed increase in Na-coupled sugar and amino acid transport induced by high NaCl diets in hen colon. A comparison was also made between the dependence of the Na-coupled transport on a pure wheat/barley/soya diet against a diet with supplements of essential amino acids, fatty acids, vitamins, and trace elements, as a test for possible elimination of the cotransporters due to a deficient diet. Na/nutrient-coupled transport was measured as changes in short circuit current. The level of expressed Na/glucose cotransporters, SGLT1, due to dietary alterations was followed by quantitative Western blot and immunodetection of SGLT1 in colon, and the dietary effects on plasma aldosterone were assessed as well. An observed switch in transport from amiloride-sensitive electrodiffusive Na transport to phlorizin-sensitive Na/D-glucose cotransport and Na/amino acid-coupled transport is caused solely by increasing Na+ in the diet. Thus, neither dietary Cl- nor the dietary supplements altered the expression of Na(+)-coupled nutrient transport processes. Corroborating these findings, only Na+ in the diet increased the expression of SGLT1 in colon epithelium and suppressed aldosterone level in plasma.

Cation effects on protein conformation and transport in the Na+/glucose cotransporter.

Cation-driven cotransporters are essential membrane proteins in procaryotes and eucaryotes, which use the energy of the transmembrane electrochemical gradient to drive transport of a substrate against its concentration gradient. Do they share a common mechanism? Cation selectivity of the rabbit isoform of the Na+/glucose cotransporter (SGLT1) was examined using the twoelectrode voltage clamp and the Xenopus oocyte expression system. The effect of H+, Li+, and Na+ on kinetics of SGLT1 was compared to the effects of these cations on the bacterial melibiose. In SGLT1, substitution of H+ or Li+ for Na+ caused a kinetic penalty in that the apparent affinity for sugar (K0.5sugar) decreased by an order of magnitude or more (from 0.2 to 30 mM) depending on the membrane potential and cation. The effect of the cation on the K0.5sugar/V profiles was independent of the sugar for glucose and alpha-methyl-beta-D-glucose; this profile was maintained for galactose in Li+ and Na+, but was 2 orders of magnitude higher in H+, but the Imax for glucose, galactose, and alpha-methyl-beta-D-glucose in a given cation were identical. Li+ supported a lower maximal rate of transport (Imax) than Na+ (approximately 80% of ImaxNa), while the Imax in H+ was higher than Na+ (>/=180% of ImaxNa). Our interpretation of these results and simulations using a six-state mathematical model, are as follows. 1) Binding of the cation causes a conformational change in the sugar binding pocket, the exact conformation being determined by the specific cation. 2) Once the sugar is bound, it is transported at a characteristic rate determined by the cation. 3) Mathematical simulations suggest that the largest contribution to the kinetic variability of both cation and sugar transport is associated with cation binding. Similarity to the effects of cation substitution in MelB suggests that the mechanism of energy coupling has been evolutionarily conserved.

Sodium cotransporters.

Recent studies of cloned mammalian sodium cotransporters in heterologous systems have revealed that these integral membrane proteins serve multiple functions as cotransporters, uniporters, channels and water transporters. Some progress has been gained in understanding their secondary structure, but information on helical bundling and tertiary structure is lacking. Site-directed mutagenesis and the construction of chimeras have resulted in the identification of residues and domains involved in ligand binding, and natural mutations have also been found that are responsible for human genetic diseases. Major factors in the short-term regulations of cotransporter function by protein kinases are exocytosis and endocytosis.

Kinetic and specificity differences between rat, human, and rabbit Na+-glucose cotransporters (SGLT-1).

The Na+ activation and substrate specificity of human, rabbit, and rat Na+-glucose cotransporter (SGLT-1) isoforms were characterized using the Xenopus oocyte expression system and the two-electrode voltageclamp method. We find that there are differences, major and minor, in both the kinetics and substrate specificities between these isoforms; the substrate concentration at half-maximal current (K0.5) for hexoses varies from 0.2 to > 40 mM, depending on the species and sugar; the affinity constant (Ki) for phlorizin, the classic competitive inhibitor of SGLT-1, varies lover two orders of magnitude (rat Ki = 0.03 microM vs. rabbit Ki = 1.4 microM); and some glucoside inhibitors of the rabbit isoform, p-nitrophenyl glucose and beta-naphthyl glucose, are transported by the human and rat transporters. Na+ activation is more sensitive to membrane potential in the human and rat isoforms compared with rabbit. The rabbit isoform has a higher apparent affinity for alpha-methylglucose and 3-O-methylglucose by a factor of two than either human or rat. These results can be quantitatively fitted by our six-state kinetic model of SGLT-1, providing insight into the processes involved in these changes. For example, the model predicts that Na+ binding (rate constant, k12) in human and rat SGLT-1 is similar but is fourfold larger than in rabbit, whereas sugar binding (k23) in rabbit and rat is similar but double the value in human SGLT-1. The differences in the primary amino acid sequences between these three homologous proteins must account for the kinetic and substrate specificity differences, and comparisons of the functional properties and amino acid sequences of SGLT-1 isoforms provide useful information about structure/function relationships.

Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.

To investigate the role of charged intramembrane residues in the function of the rabbit Na+/glucose cotransporter (rbSGLT1) we substituted arginine-427 (R427) by alanine in the putative domain M9 SGLT1. This residue is conserved in all the members of the SGLT1 family. The mutant protein (R427A) was expressed in Xenopus oocytes and, although Western blot analysis revealed that it was produced in amounts comparable to wild-type, no function was measured. Freeze-fracture analysis showed that R427A SGLT1 was not in the plasma membrane while immunocytochemical experiments localized the transporter to just beneath it. These results indicate that arginine-427 plays a critical role in SGLT1 trafficking to the plasma membrane.