Pflugers Arch - Eur J Physiol (2004) 447:510–518 DOI 10.1007/s00424-003-1063-6
The ABC of Solute Carriers
Guest Editor: Matthias A. Hediger
Ernest M. Wright · Eric Turk
The sodium/glucose cotransport family SLC5
Received: 10 January 2003 / Accepted: 28 March 2003 / Published online: 14 May 2003 Springer-Verlag 2003
Abstract 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. Keywords Cotransporters · Glucose · Iodide · Choline · Vitamins · Inositol
Brief history Crane in 1961 was the first to formulate the cotransport concept to explain active transport [7]. Specifically, he proposed that the accumulation of glucose in the intestinal epithelium across the brush border membrane was coupled to downhill Na+ transport cross the brush border. This hypothesis was rapidly tested, refined and extended encompass the active transport of a diverse range of E. M. Wright ()) · E. Turk Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1751 USA e-mail:
[email protected] Tel.: +1-310-8256905, Fax: +1-310-2065886
molecules and ions into virtually every cell type (see [64]). The first cotransporter proteins identified were the rabbit intestinal brush border Na+/glucose and Na+/proline cotransporters [55, 79]. Stimulated by the success of others in cloning membrane proteins such as channels, pumps and transporters, we set out to clone cotransporters. While we were not successful in obtaining probes (peptide sequences or antibodies) to screen cDNA libraries, we developed a new method called expression cloning [17]. This originated from our finding that Na+/ glucose transport activity was quite robust in Xenopus leavis oocytes after injection of mRNA isolated from the small intestine [18]. Our strategy for isolating the cDNA coding for the rabbit intestinal Na+/glucose cotransporter (SGLT1) was to subdivide an intestinal cDNA library, synthesize cRNA from the pools of clones, inject the cRNA into oocytes, and assay for radioactive glucose uptake. Within a few months we were able to isolate a single cDNA clone, the cRNA of which increased glucose uptake into oocytes more than 1,000-fold. Expression cloning soon became the premier method for isolating clones for other transporters, for example, the Na+/myoinositol (SMIT [29]), Na+/iodide (NIS [8]), and the members of other gene families reviewed in this volume. With cDNAs in hand, it was straightforward to screen cDNA libraries for related clones. We employed the rabbit cDNA to isolate clones for the human intestinal (SGLT1) and renal (SGLT2) Na+/glucose cotransporters [19, 77]. It is now routine to identify novel genes in genomes by in silico methods. SGLT1 (SLC5A1) is the first of more than 220 members in the SLC5 family, which currently includes 11 members from the human genome (Fig. 1) and is sometimes referred to as the sodium/substrate symporter family (SSSF TC 2.A.21) (see [26, 67] and Fig. 2). The 11 human genes (Table 1 and Fig. 1) are expressed in tissues such as the small intestine, kidney, brain, muscle and thyroid gland. Despite the homology among the proteins (21–70% amino acid identity to SGLT1) there is diversity in gene structure: in eight of the ten genes mapped the coding sequences are found in 14–15 exons (SGLT1–6,
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Typical functions
Fig. 1 Unrooted phylogenetic tree of the 11 human members of the SLC5 family of cotransporters and two other vertebrate members of known function. The alignment program CLUSTAL W (http://www.ebi.ac.uk/clustalw/) and the phylogenetic display program TreeViewPPC (http://taxonomy.zoology.gla.ac.uk/rod/ treeview.html) were used to generate the tree. Members for which transport functions have been demonstrated experimentally are shown in red. Members shown in black have yet to have their function determined. In a pairwise basic local alignment search tool (BLAST) analysis the amino acid identities relative to SGLT1 (SLC5A1) were 59% SGLT2, 70% SGLT3, 56% SGLT4, 57% SGLT5, 50% SGLT6, 55% SMIT, 24% SMVT, 24% CHT, 21% AIT, and 21% NIS (SGLT Na+/glucose transporter, SMIT Na+/myoinositol transporter, SMVT Na+/multivitamin transporter, CHT choline transporter, AIT apical iodide transporter, NIS Na+/iodide transporter)
NIS, and the Na+-dependent multivitamin transporter SMVT [12, 62, 66, 69, 76]). On the other hand the coding sequences for the choline transporter (CHT) and SMIT are contained in eight and one exons respectively [39, 47, 56]. In SGLT4–6 and SMIT there is evidence for alternative splicing ([56, 62] and E. Turk, unpublished). Functional and electron microscopic assays in heterologous expression systems demonstrate that the products of six of the genes are inserted into the plasma membrane [1, 3, 4, 8, 13, 14, 15, 17, 20, 22, 25, 27, 29, 34, 43, 47, 53, 54, 59, 72, 76, 85]. SGLT2, SGLT5 and CHT are poorly expressed in cultured cells and oocytes [1, 27, 47] and, if indeed these are inserted into the plasma membranes of the native tissues, a second protein may be required for correct insertion.
Function has been ascribed directly to 9 of the 11 human genes based on studies on heterologous expression systems, and to SLC5A10 based on the function of two “orthologs” (Table 1). As expected six proteins are cotransporters; e.g., SGLT1 is a Na+/glucose and NIS is a Na+/iodide cotransporter (e.g. [59, 74]). These are physiological functions as judged by: (1) transport measurements on the native tissues where the genes are expressed, and (2) the finding that mutations in the genes cause specific diseases, e.g. glucose-galactose malabsorption and hypothyroidism (see [9, 68]). The functions of SLC5A8 and 9 (SGLT4 and -5) are not known. Additional unexpected properties of these cotransport proteins have emerged from expression studies, for example SGLT1 and NIS behave as Na+ uniporters, water channels, urea channels and cotransporters of both urea and water [8, 13, 33, 35, 37, 53, 54, 72]. This complication is further compounded by the wide ligand specificity of the transporters [4, 10, 13, 15, 20, 22] and the expression of these genes in unexpected cells and tissues (Table 1). Finally, several genes in the family have totally surprising properties, and, in at least one case, the properties of the human genes are quite different from orthologs in other species. Human SGLT3 (hSGLT3, SLC5A4) is not a Na+/sugar transporter at all, but rather a glucose-gated ion channel expressed in muscle and neurons, whereas pig SGLT3 is a tightly coupled Na+/ glucose cotransporter [11].
Brief description and physiological implications SGLT1 is expressed primarily in the brush border membrane of mature enterocytes in the small intestine where it absorbs dietary d-glucose and d-galactose from the gut lumen [80]. Although SGLT2 is poorly expressed in COS cells and oocytes, it does appears to be a lowaffinity glucose transporter [27]. SGLT2 is only expressed in the renal cortex in rats where it is assumed to be in the brush border membrane of the S1 and S2 segments of the proximal tubule and be responsible for the reabsorption of d-glucose from the glomerular filtrate [78]. Although there is genetic evidence in support of these assumptions (see below) the absence of specific antibodies and the very low expression of SGLT2 in expression systems raise nagging uncertainties. The transport activity of SGLT2 in oocytes is less than 0.1% of that for SGLT1 in paired experiments and freeze-fracture electron microscopy demonstrates that this is due to the failure of SGLT2 to reach the plasma membrane (A. Diez-Samepdro, B.A. Hirayama, G. Zampighi, E.M. Wright, unpublished). One possible explanation is that COS cells and oocytes lack a second protein necessary for the trafficking of SGLT2 to the plasma membrane. SGLT3 does not transport glucose, but the sugar depolarizes the plasma membrane in a saturable, Na+-dependent, phlorizin-sensitive manner
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Fig. 2 An unrooted phylogenetic tree of selected prokaryote and eurkaryote members of the SGLT gene family [also referred to as the sodium/substrate symporter family (SSSF) gene family] emphasizing the SLC5 family members as well as species representing the most divergent branches of the larger SGLT family. The 45 homologs selected were aligned in CLUSTAL W, and their relationships displayed using TreeViewPPC. The members highlighted are those used later to estimate the level of retained identity at each amino acid position (see Fig.3) [Afu Archaeoglobus fulgidus (Archaea), Bha Bacillus halodurans (Gram+), Bsu Bacillus subtilis (55 kDa, Gram+), Cel Caenorhabditis elegans, Cho choline transporter, Dme Drosophila melanogaster, E59 E. coli hypothetical 59 kDa, E62 E coli hypothetical 62 kDa, Eco Escherichia coli,
Fugu Fugu rubripes, h human, Hin Haemophilus influenzae, hyp hypothetical, Lpo Limulus polyphemus (horseshoe crab), Mth Methanothermobacter thermautotrophicus (Archaea), paa phenylacetic acid transporter, Pab Pyrococcus abyssi (Archaea), Pae Pseudomonas aeruginosa, panF pantothenate transporter, Pmu Pasteurella multocida, Ppu Pseudomonas putida, putP proline transporter, rab rabbit, rna from cRNA, Sau Staphylococcus aureus (Gram+), Sce Saccharomyces cerevisiae, Sco Streptomyces coelicolor, SNST1 nucleoside transporter, SAAT1 amino acid transporter (now SGLT3), Sty Salmonella typhimurium, Syn Synechocystis (Cyanobacteria), v Vibrio parahaemolyticus, Vch V. cholerae, Vit vitamin transporter, Xau Xanthobacter autotrophicus (dhlC), Xen Xenopus laevis]
[11]. Little is known about SGLT4 and -5 except that -4 is widely expressed in the body whereas -5 is restricted to the kidney (Table 1). The rabbit ortholog of SGLT5 (RKD) does not reach the plasma membrane when expressed in oocytes (unpublished). SGLT6 is also widely expressed [62], and the rabbit and Xenopous laevis orthologs have been shown to be Na+ cotransporters [6, 45]. Each ortholog of SGLT6 transports myo-inositol (K0.5 0.1 mM for rabbit and 0.25 mM for Xenopus) and d-glucose (K0.5 30 mM for rabbit and 6 mM for Xenopus). We suggest that SGLT6 may be the elusive, low-affinity d-glucose transporter in the small intestine. SLC5A3 (SMIT) is a widely expressed Na+/myoinositol cotransporter [2], but most of the functional studies have been carried out on the dog ortholog (see [15, 29]). The K0.5 for myo-inositol is ~50 M but SMIT also transports a variety of sugars including d-glucose with low affinity. NIS, the apical iodide transporter AIT, SMVT and CHT are a group of distant relatives (Fig. 1). NIS is a Na+/ iodide cotransporter found principally in the thyroid gland where it is responsible for the accumulation of iodide
necessary for thyroid hormones T3 and T4 [9, 74]. AIT, the closest relative of NIS, is also expressed in the thyroid but is located in the apical, not the basolateral, membrane [61]. Very preliminary experiments suggest that AIT is a ClO4-sensitive I transporter involved in the exit of iodide from the thyrocyte into the lumen of the gland. SMVT is a widely distributed multivitamin/Na+ cotransporter [57, 58, 76] but there is some uncertainty about functional aspects based on preliminary electrophysiological experiments where substrates also produce outward currents under some conditions [58]. CHT is an odd member of the SLC5 family in that the gene is expressed in the central nervous system [1, 47, 48] and that Na+/choline cotransport is Cl dependent. Transport activity into intact cells is very low and, in our hands, there is little, if any, insertion of the transporter into the oocyte plasma membrane (freeze-fracture electron microscopy). Transport activity is apparently more robust in membrane vesicles prepared from whole COS-7 cells transfected with CHT-cDNA [1].
Protein name
SGLT1
SGLT2
SMIT
SGLT3
NIS
SMVT
CHT
SGLT4
SGLT5
Human gene name
SLC5A1
SLC5A2
SLC5A3
SLC5A4
SLC5A5
SLC5A6
SLC5A7
SLC5A8**
SLC5A9
Rabbit RK-D
CHT1
SAAT1
None
None
None
Aliases
C/Na+ Uniporter Na+ Channel Urea water C/Na+
I (ClO4 , SCN, NO3 , Br)
C/Na+/Cl
Glucose activated Na+ (H+) channel
Na+ (H+)
Biotin, lipoate and pantothenate Choline
C/Na+
Small Intestine>>trachea, kidney and heart; plasma membranes
C/Na+ (H+) F/Na+ (H+). Channel: urea and water C/Na+
Kidney
Brain, heart, kidney, lung and placenta; plasma membranes Spinal cord and medulla (intracellular vesicles) Small Intestine, kidney, liver, lung and brain
Small Intestine (cholinergic neurons), skeletal muscle, kidney, uterus and testis; plasma membranes Thyroid, breast, colon and ovary; plasma membranes
Brain, heart, kidney and lung; plasma membranes
Kidney cortex
Tissues distribution and cellular/subcellular expressionT
Transport type/coupling ion*
myo-inositol (glucose)
Glucose
Glucose and galactose (urea and water)
Predominant substrates
Thyroid hormonogenesisG OMIM 601843
Glucose galactose malabsorptionG OMIM 182380 Familial renal glycosuriaG OMIM 182381 Down’s syndrome? OMIM 600444
Link to disease
17p11.2
1p32
XM_064487
HCT1951464
NM _021815
NM _021095
2p23
2q12
NM_ 000453
NM_14227
19p13.2-p12
22q12.2-q12.3
NM_006933
NM_003041
16p12-p11
21q22.12
NM_000343
Sequence Accession ID
22q13.1
Human gene locus
Internal splice in Exon 14 adds either 38 or 53 aa between TMHs 13–14 Exon 7 may be spliced out deleting 26 aa between TMHs 5–6; internal splice in exon 10 means either 36 or 52 aa between TMHs 8–9; and an internal splice in exon 12 adds either 12 or 37 aa between TMHs 11–12
Splicing within, and distal to exon 2 leads to 3 transcripts (SMIT1, SMIT2 & SMIT3). SMIT2 and -3 lack the 14th TMH
Splice variants and their specific featuresS
Table 1 The SLC5 sodium glucose cotransporter family (OMIM Online Mendelian Inheritance in Man database, TMH transmembrane helix, aa amino acids)
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12q23.1 Thyroid; apical plasma membrane
C/Na+ Rabbit ortholog: myo-inositol, chiro-inositol, glucose and xylose. Xenopus laevis ortholog: myo-inositol and glucose Iodide AIT SLC5A11
KST1 Rabbit ST1 Rabbit SMIT2 SGLT6 SLC5A10
* C cotransporter; F uniporter. Function based on results obtained with heterologous expression systems ** Provisional SGLT4 exons identified by mining the Celera databases T Tissue distribution of SGLT1–6 was determined by RNAase protection assays using gene specific probes (M. Bing, E. Turk, M.G. Martin, E.M. Wright, unpublished). Also includes data from the original cloning papers and GeneCard (EST, and/or DNA array) G Gene defect S Potential alternative splice sites ([56, 62] and E. Turk, unpublished) Original references may be obtained through the Accession numbers and/or the text Note: single nucleotide polymorphisms (SNPs) and variants in the NCBI SNP database are not included.
NM_052944 16p12.1 Small Intestine, brain, kidney, liver, heart, and lung
NM_145913
Splicing eliminates exon 6 and TMH 4
Sequence Accession ID Tissues distribution and cellular/subcellular expressionT Transport type/coupling ion* Protein name Human gene name
Table 1 (continued)
Aliases
Predominant substrates
Link to disease
Human gene locus
Splice variants and their specific featuresS
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Regulation of expression Relatively little work has been published on the regulation of expression of the human members of the SLC5 family, apart from SLC5A3 which is involved intimately in osmoregulation in a variety of cells [16]. The accumulation of myo-inositol in cells is a fundamental cellular response to hypertonicity and transport is regulated the transcriptional level. Hypertonicity increases the abundance of a nuclear protein, tonicity-responsive enhancer binding protein (TonEBP), that binds to the upstream SMIT promoter element TonE, which in turn leads to increased transcription, SMIT protein levels and transport. [16]. There is some evidence for alternative splicing of the SMIT gene [56], but the physiological significance is not yet clear. Thyroid-stimulating hormone (TSH) increases NIS expression in the thyroid gland through a cGMP pathway [9]. However, the analysis of the human and rat NIS promoters have not yet reached consensus about factors controlling the tissue specific expression and regulation of NIS. In general, the study of transcriptional regulation of SLC5 family members is in its infancy (see, for example [42]). We do note that in some model systems, e.g. sheep, there is evidence for the translational regulation of transport activity [32, 63].
Biochemistry and structure All 11 genes are predicted to code for 60- to 80-kDa proteins containing 580 and 718 residues [1, 2, 19, 47, 61, 62, 65, 76, 77]. The protein secondary structure of SGLT1 is shown in Fig. 3. Secondary structure analysis suggests the presence of 14 transmembrane helices in all but NIS and AIT, which lack the 14th transmembrane helix (see [67]). The N-terminal hydrophilic loop is located on the extracellular side of the membrane and there are a variable number of consensus sites for N-linked glycosylation, but in both SGLT1 and NIS glycosylation is not required for activity. The preponderance of experimental data obtained for rabbit and human SGLT1, rat NIS and the related bacterial Na+ cotransporters for galactose (vSGLT) and proline (PutP) provides strong supporting evidence for the secondary structural models. The evidence comes from glycosylation studies, antibody tagging, and mass spectrometry (see [9, 26, 70, 71]). Freeze-fracture electron microscopy provides direct evidence that both SGLT1 and vSGLT function as 14 transmembrane helical monomers. Studies are in progress to examine the structure of recombinant proteins using mass spectrometry, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and electron microscopy [26, 31, 71, 86], but so far there are no 3D crystal structures for cotransporters. Considerable progress has been made in the understanding of how cotransporters work based on electrophysiological, biochemical, and electron microscopic studies of proteins expressed in heterologous expression
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Fig. 3 A secondary structure model for human SGLT1 illustrating levels of evolutionary retention at each amino acid residue position. There are 14 transmembrane helices and both the N- and Cterminals face the extracellular fluid [67]. The 14th transmembrane helix is absent in both NIS and AIT. The 45 homologs displayed in Fig. 2 were aligned in CLUSTAL W. Only the 19 most divergent species (those highlighted in Fig. 2) were retained in a final alignment. Residue positions with a gap in hSGLT1 were discarded, closing all hSGLT1 gaps, and for each column the sum of pairwise identities was calculated. These 664 sums were normalized to a range of 0–100% identity retention, and each amino acid position was then associated with one of five arbitrary levels
(bins) of retained identity. The levels of residue conservation in these 19 most divergent members of the large 220-member SSSF family (see Fig. 2) are indicated above by a five-color code [26, 67]. While 10 residues have very high identity conservation among pro- and eukaryote homologs, 459 residues (69%) show little or no identity conservation at all (see color code). There is generally higher conservation in the N-terminal half of the protein, which is consistent with evidence showing that SGLT1 sugar transport (and by inference the transport of the diverse substrates recognized by homologous species) occurs through a pathway formed by the Cterminal helices [10, 36, 44, 49, 50, 51, 52]
systems such as oocytes [10, 13, 15, 21, 22, 26, 34, 36, 44, 53, 54, 59, 78, 82, 85]. In general, all cotransporters or symporters, whether members of this or other gene families seem to work in the same fashion, i.e. alternating access models where the direction and rate of transport depends on the ligand concentrations on each side of the membrane and the membrane voltage. Transport occurs in an ordered manner and the maximum turnover of the transporters is in the range of 1–100/s at room temperature. Further discussion of the cotransport mechanisms is beyond the scope of this review. Nevertheless it should be pointed out that not all SLC5 members are cotransporters; e.g., SGLT3 is a glucosegated ion channel expressed in cholinergic neurons and at the neuromuscular junction [11]. In addition, cotransporters have other functions, e.g. SGLT1 and NIS behave as Na+ uniporters, water channels, urea channels and cotransporters of water and urea (see [33, 35, 37, 38, 52]).
mutations in the SGLT1 gene [28, 30, 40, 41, 68, 83, 84]. This is a rare autosomal recessive disease (Online Mendelian Inheritance in Man data base accession No. OMIM 182380) that presents in newborn infants as a lifethreatening diarrhea. The diarrhea ceases promptly on removing dietary glucose, galactose and lactose, but returns immediately on reintroducing one of the offending sugars into the child’s diet. In some 46 patients we have identified the mutations in the SGLT1 gene that cause the defect in sugar transport. These include missense, nonsense, frame-shift, and splice-site mutations. The missense mutations were studied using the oocyte expression system, where the defect in transport in 22 out of 23 mutants was due to missorting of the protein in the cell. Surprisingly, some very conservative GGM mutations cause the trafficking defect; e.g., four are alanine to valine trafficking mutants. Normal trafficking is restored when valine is replaced by cysteine, suggesting that slight conformational changes in the protein interfere with interactions between the transport vesicles containing SGLT1 and the motor proteins responsible for delivery of the vesicle to the plasma membrane. Mutations in the NIS gene have been identified as the cause of congenital iodide transport defects (OMIM
Pathology The most well known genetic disease in this family is glucose-galactose malabsorption (GGM), which involved
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601843 and see [9]). This is also a rare autosomal recessive disease and, interestingly, most of the patients reported so far are from Japan. Unlike GGM, a fair number of the patients bear the same mutation, T345P, and the defect appears to be due to a defective protein that reaches the plasma membrane. The strongest evidence to date that SGLT2 is the major transporter involved in the reabsorption of glucose from the glomerular filtrate comes from the analysis of a patient with autosomal recessive renal glycosuria [73]. DNA sequencing revealed a homozygous nonsense mutation in exon 11 of SGLT2 in the patient, and a heterozygous mutation at the same position in both parents and a younger brother. Renal glycosuria is not accompanied by GGM, but GGM patients show a mild renal glycosuria (see [83]).
Pharmacological and pharmaceutical aspects Extensive studies have been carried out on the role of protein kinases in the regulation of transport activity and this includes member of the SLC5 family. Perhaps the most surprising observation is that the kinases do not simply regulate transport by direct transporter phosphorylation. Perhaps the best example is SGLT1 where protein kinases (PKA and PKC) regulate transport by rapid insertion (or retrieval) of the transporter into the plasma membrane [23, 81]. There has only been minor interest by the pharmaceutical industry in this gene family. However, it should be noted that SGLT1 is the protein that underlies the very successful worldwide treatment of secretory diarrhea by oral rehydration therapy (ORT) [24]. It has been estimated that, annually, ORT saves the lives of more than 1,000,000 children afflicted with cholera. An interest is emerging in targeting the renal SGLT2 to control blood glucose in diabetic patients, and an oral prodrug (phlorizin analog) has been developed [46]. In rats the prodrug does not inhibit the intestinal transporter, but after absorption and activation it produces renal glycosuria and a lowering of blood glucose. What remains to be evaluated is the effect of this drug on other members of the SGLT family expressed in the body, e.g. SGLT1, SGLT3, and SMIT. There has been a report on the use of SGLTs to deliver anti-tumor drugs. This approach depends on the expression of SGLT genes in tumor cells and would take advantage of the ability of cotransporters to accumulate toxic substrates in cells. For example, a nitrogen mustard glucoside (glucosfamide), a putative substrate for SGLT3 [75], has been evaluated in a phase-1 clinical trial [5]. Finally, there is excitement in some quarters about using NIS for the diagnosis and treatment of cancers [60]. Acknowledgements Our research on SLC5 over the past two decades has been supported by grants from the National Institutes of Health (DK19567; DK44602 and DK44582) and has been made
possible by the talent of students, fellows and collaborators cited in the references.
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