Eric Turk

The sodium/glucose cotransport family SLC5

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

Intestinal absorption in health and disease--sugars.

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

Molecular basis for glucose-galactose malabsorption

Glucose-galactose malabsorption (GGM) is an autosomal recessive disease that presents in newborn infants as a life-threatening diarrhea. The diarrhea ceases within 1 h of removing oral intake of lactose, glucose, and galactose, but promptly returns with the introduction of one or more of the offending sugars into the diet. Our goal is to determine whether or not mutations in the sodium-glucose cotransporter gene (SGLT1) are responsible for GGM. We first isolated the human cDNA (hSGLT1), mapped the gene and identified its chromosomal location (22q13.1). Our approach was then to screen GGM patients for mutations in hSGLT1 and then determine if these caused defects, in sugar transport using the Xenopus laevis oocyte expression system. In 46 patients we have identified the mutations responsible for GGM. These included missense, nonsense, frame shift, splice site, and promoter mutations. In 30 patients, the same mutations were on both alleles, and the remaining 16 had different mutations on each allele (compound heterozygotes). Several mutations (e.g., C355S) were found in unrelated patients. The nonsense, frame shift, and splice site mutations all produce nonfunctional truncated proteins. In 22 out of the 23 missense mutations tested in the oocyte expression system, the proteins were translated and were stable in the cell, but did not reach the plasma membrane. In four of these mutants, an alanine residue was replaced by a valine, and in two, the trafficking defect was rescued by changing the valine to cysteine. One mutant protein (Q457R) did reach the plasma membrane, but it was unable to transport the sugar across the cell membrane. We conclude that mutations in the SGLT1 gene are the cause of glucose-galactose malabsorption, and sugar transport is impaired mainly because the mutant proteins are either truncated or are not targeted properly to the cell membrane.

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.

The Na+/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni2+ 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 μm(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.

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

The human intestinal proton‐coupled oligopeptide transporter hPEPT1 has been implicated in the absorption of pharmacologically active compounds. We have investigated the interactions between a comprehensive selection of drugs, and wild‐type and variant hPEPT1s expressed in Xenopus oocytes, using radiotracer uptake and electrophysiological methods. The 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 (Qmax) and a shift of the midpoint voltage (V0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly‐Sar > NAAG, 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.

Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects

BACKGROUND & AIMS 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Sodium cotransport proteins

Significant advances have been made in elucidating the structure of Na+ cotransport proteins. Some fifteen of these low-abundance proteins have been cloned, sequenced and functionally expressed. They are members of the 12 membrane-spanning superfamily and they segregate into two groups, the Na+/glucose (SGLT1) and Na+/Cl-/GABA (GAT-1) families. SGLT1 transporters are expressed in bacteria and animal cells, while GAT-1 transporters are mostly expressed in the brain. None have yet been found in plants.

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.

Prenatal identification of a heterozygous status in two fetuses at risk for glucose-galactose malabsorption

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 probands 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 allele and potentially decrease the postnatal morbidity.

Molecular genetics of the human Na+/glucose cotransporter.

SummaryRecent success in expression cloning has revealed the primary structure of the Na+/glucose cotransporter from rabbit small intestine, and this has subsequently led to the cloning of the Na+/glucose cotransporters from human small intestine and human kidney. Close homology is evident between the rabbit and human intestinal Na+/glucose cotransporters at the DNA level, and the predicted amino acid and secondary structure levels. The Na+/glucose cotransporter amino acid sequence from human kidney is 57% identical with that from human small intestine. Significant homology also exists between these Na+/glucose cotransporters and theE. coli Na+/proline cotransporter (putP). The rabbit intestinal Na+/glucose cotransporter has 11 potential membrane spanning regions and 2 hydrophilic regions containing highly charged residues. The amino acid sequence shows two potential N-glycosylation sites (N-X-T/S). Using an in vitro translation approach we were able to determine that only one of these (Asn 248) is glycosylated. Expression experiments withXenopus oocytes using the N-glycosylation inhibitor tunicamycin indicate that glycosylation of Asn 248 is required for functional expression of the transporter. The N-X-T/S sequence at Asn 248 is conserved in the human intestinal and the human renal Na+/glucose cotransporter. Chromosomal localization studies map the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2→qter region of chromosome 22 and the human renal Na+/glucose cotransporter gene (SGLT2) to the q-arm of chromosome 16. Thus the intestinal and renal Na+/glucose cotransporters are encoded by different genes located on different chromosomes. This is consistent with the observation that inherited defects of the transporters, intestinal glucose/galactose malabsorption and renal glycosuria, do not appear to be genetically linked.