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Papers by Eric Turk
Gastroenterology Clinics of North America, 1995
In this article, genetic disorders of the gastrointestinal and hepatobiliary tracts ranging from ... more In this article, genetic disorders of the gastrointestinal and hepatobiliary tracts ranging from common to very rare are reviewed. The first portion of each section highlights the clinical manifestations of the disorder; the second and more detailed portion of each section discusses what is currently known about the biology and genetics of the disorder. The potential use of gene therapy to treat many of these disorders is also reviewed.
Journal of Biological Chemistry, 1994
Intestinal uptake of dietary glucose and galactose is mediated by the SGLTl Na+/glucose cotranspo... more Intestinal uptake of dietary glucose and galactose is mediated by the SGLTl Na+/glucose cotransporter of the brush border. An SGLTl 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 SGLTl Na+/glucose cotransporter gene from cosmid and A 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. SGLTl 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. The primary protein carrier responsible for the uptake of the dietary sugars glucose and galactose from the intestinal lumen is the Na+/glucose cotransporter encoded by the gene SGLTl(1, 2). The 84-kDa glycoprotein of 12 postulated membrane spans is localized i n the brush border of the intestinal epithelium and actively imports lumenal sugar against the enterocyte transmembrane concentration gradient by coupling sugar transport with that of Na' down its electrochemical gradient (1,3). Northern blots of several organs indicate that SGLTl mRNA transcription is mostly restricted to intestine and kidney (4). Exit of sugar from the enterocyte cytosol to blood is completed by the facilitated glucose carrier GLUT2 localized in the basolateral membrane (1). The facilitated transporters belong to a distinct protein family with no homology to SGLTl(5). Members of the SGLTl cotransporter family, which includes the mammalian Na+/nucleoside and Na+/myoinositol transporters, as well as the bacterial Na+/pantothenate and Na+/proline transporters (6), are predicted to incorporate 12-membrane spans, as are those of the GLUT family.
Acta physiologica Scandinavica. Supplementum, 1998
Cotransporters are a major class of membrane transport proteins that are responsible for the accu... more 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 ...
Genomics, 1993
The Na[sup +]/glucose cotransporter gene SGLT1 encodes the primary carrier protein responsible fo... more The Na[sup +]/glucose cotransporter gene SGLT1 encodes the primary carrier protein responsible for the uptake of the dietary sugars glucose and galactose from the intestinal lumen. SGLT1 transport activity is currently exploited in oral rehydration therapy. The 75-kDa glycoprotein is localized in the brush border of the intestinal epithelium and is predicted to comprise 12 membrane spans. In two patients
European Journal of Biochemistry, 1988
Physiology of the Gastrointestinal Tract, 2006
The Journal of Physiology, 2006
The human intestinal proton-coupled oligopeptide transporter hPEPT1 has been implicated in the ab... more 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 β-lactam antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil, the antineoplastics δ-aminolevulinic acid (δ-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 (Q max) and a shift of the midpoint voltage (V 0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly-Sar > NAAG, δ-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.
Proceedings of the National Academy of Sciences, 1998
The mechanism by which cotransport proteins couple their substrates across cell membranes is not ... more 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 sh...
Prenatal Diagnosis, 1996
Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder which presents with seve... more Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder which presents with severe osmotic diarrhoea shortly after birth. Two proband siblings with GGM were previously demonstrated to contain a missense mutation (D28N) in the Na(+)-dependent glucose/galactose cotransporter (SGLT1) that accounts for the defect in sugar absorption. Prenatal screening for GGM was performed in two subsequent pregnancies in this large consanguineous family. The first exon of the SGLT1 gene was PCR-amplified from genomic DNA and screened for the presence of the D28N mutation by EcoRV restriction digestion. The proband's sibling was heterozygous and a cousin was not a carrier of the D28N mutation. Both children at 2-years of age remain healthy and have had no diarrhoeal symptoms. Molecular biology techniques will allow a prospective determination of the presence of an abnormal SGLT1 allete and potentially decrease the postnatal morbidity.
Physiology, 2004
SLC5 is an ancient gene family with 11 members in the human genome. These membrane proteins have ... more 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.
Pfl�gers Archiv European Journal of Physiology, 2004
Journal of Molecular Biology, 2003
Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber c... more 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. Twodimensional 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 (,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.
Journal of Biological Chemistry, 2000
The Na ؉ /galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexa... more The Na ؉ /galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni 2؉ affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na ؉-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K m values of 129 mM (Na ؉) and 158 M (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters. Members of the Na ϩ-coupled cotransporter family SGLT (1, 2) of intramembrane proteins occur in the Bacteria, Archaea, and Eucarya. Substrates range from sugars to the proline zwitterion and the anions pantothenate, biotin, and iodide (3-10). SGLT members share in common the membrane topological motifs of an externally oriented N terminus, and at least 13 transmembrane spans (2), features that have been verified in the human SGLT1 Na ϩ /glucose (11), the bacterial putP Na ϩ / proline (12), and the rat NIS Na ϩ /iodide cotransporters (13). The mammalian Na ϩ /glucose cotransporter SGLT1 couples
Journal of Biological Chemistry, 1997
To test the hypothesis that the C-terminal half of the Na ؉ /glucose cotransporter (SGLT1) contai... more To test the hypothesis that the C-terminal half of the Na ؉ /glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C 5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C 5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C 5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C 5-cRNA injection compared with noninjected oocytes. The diameters of the C 5 particles were heterogeneous (4.8 ؎ 0.3, 7.1 ؎ 1.2, and 10.3 ؎ 0.8 nm) in contrast to the endogenous particles (7.6 ؎ 1.2 nm). C 5 increased the ␣-methyl-D-glucopyranoside (␣MDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K 0.5 ␣MDG of 50 mM and a turnover of ϳ660 s ؊1. Influx was independent of Na ؉ with transport characteristics similar to those of SGLT1 in the absence of Na ؉ : 1) selective (␣MDG > D-glucose > D-galactose > > L-glucose Ϸ D-mannose), 2) inhibited by phloretin, K i PT ؍ ϳ500 M, and 3) insensitive to phlorizin. These results indicate that C 5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC 5 (the human equivalent of C 5), hC 4 (human SGLT1 amino acids 407-648, helices 10-13), and hN 13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.
Journal of Biological Chemistry, 1996
Gastroenterology, 2000
ABSTRACT The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epith... more ABSTRACT The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.
Gastroenterology, 1997
Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose... more Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose-galactose malabsorption, characterized by severe diarrhea. This study focused on a unique proband with glucose-galactose malabsorption who was investigated 30 years ago, and the aims of the study were to identify mutations in the SGLT1 gene and to determine the defect in sugar transport. Mutations were identified by sequencing, and each mutant protein was then studied using a Xenopus oocyte heterologous expression system. Analysis included Western, freeze fracture, radiotracer uptake, and electrophysiological assays. Two heterozygous missense mutations (Cys355Ser and Leu147Arg) were identified that entirely eliminated Na+/sugar cotransport activity. Western blot analysis showed that the levels of both mutant proteins in the oocyte were comparable to wild-type SGLT1, but no complex glycosylation was detected. No SGLT1 charge movements were observed with the mutant proteins, and freeze fracture data showed that neither mutant protein reached the plasma membrane. The Cys355Ser and Leu147Arg mutations eliminate the Na+/sugar cotransport by blocking the transfer of SGLT1 protein from the endoplasmic reticulum to the plasma membrane. This is consistent with earlier studies on phlorizin binding to the brush border membrane of duodenal biopsy specimens from this patient.
Current Opinion in Cell Biology, 1996
Recent studies of cloned mammalian sodium cotransporters in heterologous systems have revealed th... more 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 regulation of cotransporter function by protein kinases are exocytosis and endocytosis.
Gastroenterology Clinics of North America, 1995
In this article, genetic disorders of the gastrointestinal and hepatobiliary tracts ranging from ... more In this article, genetic disorders of the gastrointestinal and hepatobiliary tracts ranging from common to very rare are reviewed. The first portion of each section highlights the clinical manifestations of the disorder; the second and more detailed portion of each section discusses what is currently known about the biology and genetics of the disorder. The potential use of gene therapy to treat many of these disorders is also reviewed.
Journal of Biological Chemistry, 1994
Intestinal uptake of dietary glucose and galactose is mediated by the SGLTl Na+/glucose cotranspo... more Intestinal uptake of dietary glucose and galactose is mediated by the SGLTl Na+/glucose cotransporter of the brush border. An SGLTl 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 SGLTl Na+/glucose cotransporter gene from cosmid and A 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. SGLTl 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. The primary protein carrier responsible for the uptake of the dietary sugars glucose and galactose from the intestinal lumen is the Na+/glucose cotransporter encoded by the gene SGLTl(1, 2). The 84-kDa glycoprotein of 12 postulated membrane spans is localized i n the brush border of the intestinal epithelium and actively imports lumenal sugar against the enterocyte transmembrane concentration gradient by coupling sugar transport with that of Na' down its electrochemical gradient (1,3). Northern blots of several organs indicate that SGLTl mRNA transcription is mostly restricted to intestine and kidney (4). Exit of sugar from the enterocyte cytosol to blood is completed by the facilitated glucose carrier GLUT2 localized in the basolateral membrane (1). The facilitated transporters belong to a distinct protein family with no homology to SGLTl(5). Members of the SGLTl cotransporter family, which includes the mammalian Na+/nucleoside and Na+/myoinositol transporters, as well as the bacterial Na+/pantothenate and Na+/proline transporters (6), are predicted to incorporate 12-membrane spans, as are those of the GLUT family.
Acta physiologica Scandinavica. Supplementum, 1998
Cotransporters are a major class of membrane transport proteins that are responsible for the accu... more 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 ...
Genomics, 1993
The Na[sup +]/glucose cotransporter gene SGLT1 encodes the primary carrier protein responsible fo... more The Na[sup +]/glucose cotransporter gene SGLT1 encodes the primary carrier protein responsible for the uptake of the dietary sugars glucose and galactose from the intestinal lumen. SGLT1 transport activity is currently exploited in oral rehydration therapy. The 75-kDa glycoprotein is localized in the brush border of the intestinal epithelium and is predicted to comprise 12 membrane spans. In two patients
European Journal of Biochemistry, 1988
Physiology of the Gastrointestinal Tract, 2006
The Journal of Physiology, 2006
The human intestinal proton-coupled oligopeptide transporter hPEPT1 has been implicated in the ab... more 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 β-lactam antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil, the antineoplastics δ-aminolevulinic acid (δ-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 (Q max) and a shift of the midpoint voltage (V 0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly-Sar > NAAG, δ-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.
Proceedings of the National Academy of Sciences, 1998
The mechanism by which cotransport proteins couple their substrates across cell membranes is not ... more 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 sh...
Prenatal Diagnosis, 1996
Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder which presents with seve... more Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder which presents with severe osmotic diarrhoea shortly after birth. Two proband siblings with GGM were previously demonstrated to contain a missense mutation (D28N) in the Na(+)-dependent glucose/galactose cotransporter (SGLT1) that accounts for the defect in sugar absorption. Prenatal screening for GGM was performed in two subsequent pregnancies in this large consanguineous family. The first exon of the SGLT1 gene was PCR-amplified from genomic DNA and screened for the presence of the D28N mutation by EcoRV restriction digestion. The proband's sibling was heterozygous and a cousin was not a carrier of the D28N mutation. Both children at 2-years of age remain healthy and have had no diarrhoeal symptoms. Molecular biology techniques will allow a prospective determination of the presence of an abnormal SGLT1 allete and potentially decrease the postnatal morbidity.
Physiology, 2004
SLC5 is an ancient gene family with 11 members in the human genome. These membrane proteins have ... more 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.
Pfl�gers Archiv European Journal of Physiology, 2004
Journal of Molecular Biology, 2003
Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber c... more 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. Twodimensional 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 (,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.
Journal of Biological Chemistry, 2000
The Na ؉ /galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexa... more The Na ؉ /galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni 2؉ affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na ؉-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K m values of 129 mM (Na ؉) and 158 M (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters. Members of the Na ϩ-coupled cotransporter family SGLT (1, 2) of intramembrane proteins occur in the Bacteria, Archaea, and Eucarya. Substrates range from sugars to the proline zwitterion and the anions pantothenate, biotin, and iodide (3-10). SGLT members share in common the membrane topological motifs of an externally oriented N terminus, and at least 13 transmembrane spans (2), features that have been verified in the human SGLT1 Na ϩ /glucose (11), the bacterial putP Na ϩ / proline (12), and the rat NIS Na ϩ /iodide cotransporters (13). The mammalian Na ϩ /glucose cotransporter SGLT1 couples
Journal of Biological Chemistry, 1997
To test the hypothesis that the C-terminal half of the Na ؉ /glucose cotransporter (SGLT1) contai... more To test the hypothesis that the C-terminal half of the Na ؉ /glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C 5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C 5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C 5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C 5-cRNA injection compared with noninjected oocytes. The diameters of the C 5 particles were heterogeneous (4.8 ؎ 0.3, 7.1 ؎ 1.2, and 10.3 ؎ 0.8 nm) in contrast to the endogenous particles (7.6 ؎ 1.2 nm). C 5 increased the ␣-methyl-D-glucopyranoside (␣MDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K 0.5 ␣MDG of 50 mM and a turnover of ϳ660 s ؊1. Influx was independent of Na ؉ with transport characteristics similar to those of SGLT1 in the absence of Na ؉ : 1) selective (␣MDG > D-glucose > D-galactose > > L-glucose Ϸ D-mannose), 2) inhibited by phloretin, K i PT ؍ ϳ500 M, and 3) insensitive to phlorizin. These results indicate that C 5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC 5 (the human equivalent of C 5), hC 4 (human SGLT1 amino acids 407-648, helices 10-13), and hN 13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.
Journal of Biological Chemistry, 1996
Gastroenterology, 2000
ABSTRACT The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epith... more ABSTRACT The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.
Gastroenterology, 1997
Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose... more Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose-galactose malabsorption, characterized by severe diarrhea. This study focused on a unique proband with glucose-galactose malabsorption who was investigated 30 years ago, and the aims of the study were to identify mutations in the SGLT1 gene and to determine the defect in sugar transport. Mutations were identified by sequencing, and each mutant protein was then studied using a Xenopus oocyte heterologous expression system. Analysis included Western, freeze fracture, radiotracer uptake, and electrophysiological assays. Two heterozygous missense mutations (Cys355Ser and Leu147Arg) were identified that entirely eliminated Na+/sugar cotransport activity. Western blot analysis showed that the levels of both mutant proteins in the oocyte were comparable to wild-type SGLT1, but no complex glycosylation was detected. No SGLT1 charge movements were observed with the mutant proteins, and freeze fracture data showed that neither mutant protein reached the plasma membrane. The Cys355Ser and Leu147Arg mutations eliminate the Na+/sugar cotransport by blocking the transfer of SGLT1 protein from the endoplasmic reticulum to the plasma membrane. This is consistent with earlier studies on phlorizin binding to the brush border membrane of duodenal biopsy specimens from this patient.
Current Opinion in Cell Biology, 1996
Recent studies of cloned mammalian sodium cotransporters in heterologous systems have revealed th... more 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 regulation of cotransporter function by protein kinases are exocytosis and endocytosis.