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E M Wright

Publications and source records attributed to E M Wright.

At least 109 records · Page 6Linked to original sources
PubMed1996

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.

3-O-Methylglucose↗
PubMed1995

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.

Animals↗
PubMed1995

Kinetics of steady-state currents and charge movements associated with the rat Na+/glucose cotransporter.

The rat Na+/glucose cotransporter (SGLT1) was expressed in Xenopus oocytes and steady-state and transient currents were measured using a two-electrode voltage clamp. The maximal glucose induced Na(+)-dependent inward current was approximately 300-500 nA. The apparent affinity constants for sugar (alpha-methyl-D-glucopyranoside; alpha MDG) (K alpha MDG 0.5) and sodium (KNa0.5) at a membrane potential of -150 mV were 0.2 mM and 4 mM. The KNa0.5 increased continuously with depolarizing potentials reaching 40 mM at -30 mV, K alpha MDG 0.5 was steeply voltage dependent, 0.46 mM at -30 mV and 1 mM at -10 mV. From all tested monovalent cations only Li+ could substitute for Na+, but with lower affinity. The relative substrate specificity was D-glucose > alpha MDG approximately D-galactose > 3-O-Me-Glc >> beta-naphthyl-D-glucoside >> uridine. Phlorizin (Pz), the specific blocker of sugar transport, showed an extremely high affinity for the rat cotransporter with an inhibitor constant (KPzi) of 12 nM. SGLT1 charge movements in the absence of sugar were fitted by the Boltzmann equation with an apparent valence of the movable charge of approximately 1, a potential for 50% maximal charge transfer (V0.5) of -43 mV, and a maximal charge (Qmax) of 9 nanocoulombs. The apparent turnover number for the rat SGLT1 was 30 s-1. Model simulations showed that the kinetics of the rat SGLT1 are described by a six-state ordered nonrapid equilibrium model, and comparison of the kinetics of the rat, rabbit and human cotransporters indicate that they differ mainly in their presteady-state kinetic parameters.

Amino Acid Sequence↗
PubMed1995

Sequence and expression of Sox-18 encoding a new HMG-box transcription factor.

The newly identified Sox gene family (Sry-like HMG-box gene) is characterized by a conserved DNA sequence encoding a domain of approx. 80 amino acids (aa) which is responsible for sequence-specific DNA binding. The first member isolated, the mammalian Y-linked testis-determining gene, Sry, is necessary and sufficient for male development. We report here the identification of two new members of this family, Sox-17 and 18. We have determined the full cDNA sequence of Sox-18 which encodes a protein of 378 aa. Sox-18 mRNA transcripts were restricted to heart, lung and skeletal muscle in the adult mouse.

Amino Acid Sequence↗
PubMed1995

Neurotrophic factors. Neurotrophin autocrine loops.

Developing neurons depend on neurotrophins supplied by the tissues they innervate. Before and after this period of target-dependent survival, brain-derived neurotrophic factor also has autocrine actions on some neurons.

Animals↗
PubMed1995

A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes.

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212 +/- 48 particles/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) only on the P face. The utility and generality of the method were demonstrated by estimating the "gating charge" per particle of the Na+/glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The "gating charges" were 3-fold larger than the "effective valences" calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

Animals↗
PubMed1995

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes.

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (Vm), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: KMI0.5, KNa0.5, and the Hill coefficient n. At 100 mM NaCl, KMI0.5 was about 50 microM and was independent of Vm. At 0.5 mM myo-inositol, KNa0.5 ranged from 76 mM at Vm = -50 mV to 40 mM at Vm = -150 mV. n was voltage independent with a value of 1.9 +/- 0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent KI of 64 microM at Vm = -50 mV and 130 microM at Vm = -150 mV. To examine sugar specificity, sugar-induced steady-state currents (at Vm = -150 mV) were recorded for a series of sugars, each at an external concentration of 50 mM. The substrate selectivity series was myo-inositol, scylloinositol > L-fucose > L-xylose > L-glucose, D-glucose, alpha-methyl-D-glucopyranoside > D-galactose, D-fucose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose > D-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at Vm = -150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: D-glucose, alpha-methyl-D-glucopyranoside, D-galactose, D-fucose, 3-O-methyl-D-glucose > D-xylose, L-xylose, 2-deoxy-D-glucose > myo-inositol, L-glucose, L-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na(+)-dependent uptake of 3H-D-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mM Na+ that was abolished by phlorizin (0.5 mM). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in Vm. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.

Animals↗
PubMed1994

SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation.

Recently a member of the Na+/glucose (SGLT1) gene family of cotransporters was isolated from a pig renal cell line and was thought to be the neutral amino acid transporter System A. This cDNA (Kong, C. T., Yet, S. F., and Lever, J. E. (1993) J. Biol. Chem. 268, 1509-1512) encodes a 660-amino acid protein with 76% identity to SGLT1. To confirm and extend the kinetic characterization of SAAT1, we have expressed this clone in Xenopus oocytes and measured transport using both radiotracer and electrophysiological techniques. SAAT1 did not stimulate either 50 microM 2-(methylamino)isobutyrate uptake or 2-(methylamino)isobutyrate-evoked inward Na+ currents, but instead stimulated 50 microM alpha MG (alpha-methyl-D-glucopyranoside) uptake 27-fold from 2 +/- 1 pmol.h-1/oocyte (n = 9) to 55 +/- 6 pmol.h-1/oocyte (n = 9) and alpha MG-evoked inward Na+ currents (I) by up to 1000 nA/oocyte. The apparent affinity constant for alpha MG (K alpha MG 0.5) was approximately 2 mM and was independent of membrane potential from -30 to -150 mV but was voltage-sensitive between -30 and +30 mV. The relative sugar specificity for the transporter was alpha MG > or = D-glucose >> D-galactose >>> 3-O-methyl-D-glucopyranose, L-glucose. The sugar-evoked currents were Na(+)-dependent (KNa 0.5 approximately 10 mM at -50 mV) and the Hill coefficient was 1. KNa 0.5 decreased with hyperpolarization of the membrane from -50 to -150 mV. Phlorizin inhibited the alpha MG-evoked current with apparent Ki of 18 microM at -50 mV. We conclude that the SAAT1 cDNA encodes a renal low affinity Na+(1)/glucose(1) cotransporter and propose that pig SAAT1 be renamed pSGLT2.

Amino Acid Transport Systems↗
PubMed1994

Protons drive sugar transport through the Na+/glucose cotransporter (SGLT1).

Na(+)-dependent transporters, such as the Na+/glucose cotransporter (SGLT1) have long been characterized as having an overwhelming preference for Na+ as the essential ion. We have applied electrophysiological measurements to analyze the ability of protons to transport glucose with a cloned transporter, SGLT1, expressed in Xenopus laevis oocytes. Our results show that protons can drive sugar transport through SGLT1 in the absence of Na+ with the following characteristics: 1) the affinity of SGLT1 for H+ is 3 orders of magnitude higher than its affinity for Na+ (3 microM versus 20 mM); 2) H+ supports a higher maximum transport than Na+, suggesting an alteration in rate-limiting processes; and 3) the cation determines the transporter's affinity for sugar (at Vm = -50 mV, the apparent affinity for alpha-methyl-D-glucoside is 0.2 mM in Na+ and 20 mM in H+). The similarity in the kinetics of H(+)- and Na(+)-dependent sugar transport suggests that the transport mechanism for SGLT1 does not depend on the driving ion.

Animals↗
PubMed1994

Sodium/D-glucose cotransporter charge movements involve polar residues.

Na(+)-dependent glucose transporters (SGLT1) exhibit transient carrier currents with a time constant (tau) of 2-20 ms, and the charge transfer (Q) fits the Boltzmann equation. There is a 60-mV negative displacement in the tau/V and Q/V curves between the human and rabbit SGLT1 proteins, and the initial goal was to identify the charges responsible for these differences in kinetics. We have focused on residue 176 in putative transmembrane helix (M4) because this is an aspartic acid in rabbit and asparagine in human. Asp-176 in rabbit SGLT1 was replaced with asparagine and alanine residues, and the wild-type and mutant proteins were expressed in Xenopus laevis oocytes. A two-electrode voltage clamp was used to measure the kinetics of charge transfer. There was no difference between the wild-type and D176N, but there was a 60-mV negative shift in the tau/V and Q/V curves with D176A. This suggests that polar residues at position 176 play an important role in determining charge transfer, probably by electrostatic bonding to a neighboring polar residue in the membrane domain of the protein. The similarity between rabbit SGLT1 and the D176N mutant further indicates that other membrane residues account for the difference between rabbit and human SGLT1. There were only modest changes in the steady-state Na+/glucose cotransport kinetics between wild-type and D176A mutant transporters in the voltage range +50 to -50 mV. Model simulations show that the mutation alters the rate constants for conformational changes of the unloaded transporter. Phlorizin, a specific competitive inhibitor of sugar transport, has a lower affinity for the D176A mutant than for SGLT1. This indicates that polar residues at position 176 hydrogen bond with the -OH group on the B-phenyl ring of the inhibitor.

Amino Acid Sequence↗
PubMed1994

Steady-state and presteady-state kinetics of the H+/hexose cotransporter (STP1) from Arabidopsis thaliana expressed in Xenopus oocytes.

We have investigated the steady-state and presteady-state kinetics of the cloned H+/hexose cotransporter from Arabidopsis thaliana (STP1) expressed in Xenopus oocytes using the two-electrode voltage-clamp method. Steady-state sugar-dependent currents were measured between -150 and +50 mV as a function of external [3-O-methyl-D-glucose] (3OMG) and [H+]. At pH 6.5 (316 nM H+) the maximal current for sugar, i3OMGmax, was voltage-dependent, increasing from 40 nA at -30 mV to 95 nA at -150 mV. The apparent affinity of sugar, K3OMG0.5, at pH 6.5 decreased from 30 microM at -30 mV to 11 microM at -70 mV and was then voltage-independent between -70 and -150 mV. Increasing the extracellular [H+] to 3160 nM (pH 5.5) increased i3OMGmax to 65 nA at -30 mV and 212 nA at -150 mV. K3OMG0.5 at pH 5.5 also increased and was voltage-dependent: 15 microM at -50 mV rising to 25 microM at -150 mV. At pH 6.5 and 5.5, the Hill coefficient n for 3OMG was voltage-independent and averaged 1.2 and 1.4, respectively. At saturating [3OMG] iHmax was voltage-dependent and KH0.5 was voltage-independent, averaging 370 nM (pH 6.4). The Hill coefficient n for H+ was voltage-independent and averaged 1. The sugar specificity of STP1 was D-mannose > or = 2-deoxyglucose > D-galactose > or = 3OMG > D-xylose > D-glucose > D-fucose > D-fructose > L-glucose > L-arabinose > D-arabinose, demonstrating that STP1 has a low substrate specificity. Transient currents recorded after rapid steps in membrane potential relaxed with time constants tau, between 3 and 14 ms. The charge movement Q (the integral of the current transients) fitted to a Boltzmann relation with maximal charge Qmax of 3.4 nanocoulombs and an apparent valence z approximately 1 corresponding to a transporter density of 2 x 10(10)/oocyte. Potential for 50% Qmax (V0.5) was -28 mV. At saturating 3OMG and at low external [H+] (pH 7.5), the transient STP1 currents were eliminated. The presteady-state data indicate that STP1 can bind H+ in the absence of sugar, and the steady-state data suggest that H+/hexose cotransport occurs via a sequential mechanism.

Animals↗
PubMed1994

Transport of cycasin by the intestinal Na+/glucose cotransporter.

The medicinal and food use of seed from the cycad plant (Cycas spp.), which contains the neurotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Cycasin, the beta-D-glucoside of methylazoxymethanol might enter neurons and other cells via a glucose transporter. Since the intestinal brush-border Na+/glucose cotransporter plays a major role in the absorption of monosaccharides, the following studies were conducted to determine if cycasin, the beta-D-glucoside of methylazoxymethanol, is a substrate for the transporter. We measured the ability of cycasin to (i) inhibit Na+/glucose uptake into rabbit intestinal brush-border membrane vesicles, and (ii) to generate current by the cloned Na+/glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes. The results show that cycasin inhibits Na(+)-dependent sugar transport in the vesicles, and cycasin generates phlorizin-sensitive currents in oocytes. We conclude that cycasin is a substrate for the intestinal brush-border Na+/glucose cotransporter, albeit with a lower affinity than D-glucose. This suggests that cycasin may be absorbed from the gut lumen by the cotransporter, and as a result either cycasin or the aglycone is presented to the blood-brain barrier for uptake into the brain.

Animals↗
PubMed1994

Structure of the human Na+/glucose cotransporter gene SGLT1.

Intestinal uptake of dietary glucose and galactose is mediated by the SGLT1 Na+/glucose cotransporter of the brush border. An SGLT1 missense mutation underlies hereditary glucose/galactose malabsorption, characterized by potentially fatal diarrhea; conversely, oral rehydration therapy exploits normal transport to alleviate life-threatening diarrhea of infectious origin. We have mapped the entire human SGLT1 Na+/glucose cotransporter gene from cosmid and lambda phage clones representing a genomic region of 112 kilobases. Transcription initiation occurred from a site 27 base pairs 3' of a TATAA sequence. All exon-flanking regions were sequenced, and the entire 112-kilobase region mapped with four restriction enzymes. SGLT1 is comprised of 15 exons (spanning 72 kilobases); a possible evolutionary origin from a six-membrane-span ancestral precursor via a gene duplication event is suggested from comparison of exons against protein secondary structure and from sequence considerations. A new missense mutation in exon 1 causing glucose/galactose malabsorption is also described. This is the first Na(+)-dependent cotransporter gene structure reported. These data facilitate the search for new glucose/galactose malabsorption-related mutations in this important gene and provide a basis for future evolutionary comparisons with other Na(+)-dependent cotransporters.

Amino Acid Sequence↗
PubMed1994

Phenylglucosides and the Na+/glucose cotransporter (SGLT1): analysis of interactions.

Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na(+)-dependent uptake of 14C-alpha-methyl-D-glucopyranoside (alpha MDG) with apparent Kis in the range 0.1 to 20 mM, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na(+)-loaded transporter. Computational chemistry indicated that all these beta-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the -NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.

Animals↗
PubMed1994

An assessment of prilocaine as a topical anaesthetic agent for fibreoptic bronchoscopy in comparison with lidocaine.

We have evaluated prilocaine as a topical anaesthetic agent for fibreoptic bronchoscopy in comparison with lidocaine in terms of efficacy and safety. Forty patients were included in a randomised double-blind parallel-group study. Efficacy was assessed using visual analogue scales, a patient ranking scale and the number of doses of local anaesthetic and intravenous sedative required. Measures of toxicity included peak plasma concentration of local anaesthetic, whether supplementary oxygen was needed and change in methaemoglobin concentration. For most of the outcome variables, the medians and quartiles were similar for the two local anaesthetics. However, the median peak plasma concentration of prilocaine (0.5 micrograms.ml-1) was less than one-third that of lidocaine (1.76 micrograms.ml-1). The merits and hazards of using multiple-regression modelling to improve the precision of the analysis of the results are considered. We conclude that prilocaine can be used successfully as a topical anaesthetic agent for fibreoptic bronchoscopy and is associated with a lower risk of toxicity.

Adult↗
PubMed1994

'Active' sugar transport in eukaryotes.

Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗