Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.

rkST1, an orphan cDNA of the SLC5 family (43% identical in sequence to the sodium myo-inositol cotransporter SMIT), was expressed in Xenopus laevis oocytes that were subsequently voltage-clamped and exposed to likely substrates. Whereas superfusion with glucose and other sugars produced a small inward current, the largest current was observed with myo-inositol. The expressed protein, which we have named SMIT2, cotransports myo-inositol with a K m of 120 M and displays a current-voltage relationship similar to that seen with SMIT (now called SMIT1). The transport is Na ؉ -dependent, with a K m of 13 mM. SMIT2 exhibits phlorizin-inhibitable presteady-state currents and substrate-independent "Na ؉ leak" currents similar to those of related cotransporters. The steady-state cotransport current is also phlorizin-inhibitable with a K i of 76 M. SMIT2 exhibits stereospecific cotransport of both D-glucose and D-xylose but does not transport fucose. In addition, SMIT2 (but not SMIT1) transports D-chiro-inositol. Based on previous publications, the tissue distribution of SMIT2 is different from that of SMIT1, and the existence of this second cotransporter may explain much of the heterogeneity that has been reported for inositol transport.The first members of the vertebrate cotransporter protein family SLC5, which includes the high affinity Na ϩ /glucose cotransporter (SGLT1) and the Na ϩ /myo-inositol cotransporter (SMIT), were isolated over a decade ago based on expression of the proteins in Xenopus laevis oocytes (1, 2). Although substrates as diverse as proline, iodide, and vitamins (3) are transported by this family of proteins, the best characterized transporters remain SGLT1 and SMIT. There are also several "orphan" transporters whose cDNA has been cloned either by using labeled cDNA from members of the SLC5 family as biochemical probes or by comparing SLC5 sequence information in silico to data stored in DNA data bases (3); the newly discovered sequences are orphans in that they have no known function. Some of the orphan protein sequences are particularly similar to the protein sequences for SGLT1 and SMIT (4, 5) and presumably transport substrates similar or identical to either glucose or its isomer myo-inositol. The SLC5 proteins with known functions have generally been studied by voltageclamp experiments because these proteins are electrogenic. Also, presteady-state currents are associated with expression of these proteins at the cell surface, and some (but not all, e.g.

The orphan cotransport protein expressed by the SLC5A8 gene has been shown to play a role in controlling the growth of colon cancers, and the silencing of this gene is a common and early event in human colon neoplasia. We expressed this protein in Xenopus laevis oocytes and have found that it transports small monocarboxylic acids. The electrogenic activity of the cotransporter, which we have named SMCT (sodium monocarboxylate transporter), was dependent on external Na + and was compatible with a 3 : 1 stoichiometry between Na + and monocarboxylates. A portion of the SMCT-mediated current was also Cl − dependent, but Cl − was not cotransported. SMCT transports a variety of monocarboxylates (similar to unrelated monocarboxylate transport proteins) and most transported monocarboxylates demonstrated K m values near 100 µM, apart from acetate and D-lactate, for which the protein showed less affinity. SMCT was strongly inhibited by 1 mM probenecid or ibuprofen. In the absence of external substrate, a Na + -independent leak current was also observed to pass through SMCT. SMCT activity was strongly inhibited after prolonged exposure to high external concentrations of monocarboxylates. The transport of monocarboxylates in anionic form was confirmed by the observation of a concomitant alkalinization of the cytosol. SMCT, being expressed in colon and kidney, represents a novel means by which Na + , short-chain fatty acids and other monocarboxylates are transported in these tissues. The significance of a Na + -monocarboxylate transporter to colon cancer presumably stems from the transport of butyrate, which is well known for having anti-proliferative and apoptosis-inducing activity in colon epithelial cells.

We describe two siblings from a consanguineous family with autosomal recessive Fanconi's syndrome and hypophosphatemic rickets. Genetic analysis revealed a homozygous in-frame duplication of 21 bp in SLC34A1, which encodes the renal sodium-inorganic phosphate cotransporter NaPi-IIa, as the causative mutation. Functional studies in Xenopus laevis oocytes and in opossum kidney cells indicated complete loss of function of the mutant NaPi-IIa, resulting from failure of the transporter to reach the plasma membrane. These findings show that disruption of the human NaPi-IIa profoundly impairs overall renal phosphate reabsorption and proximal-tubule function and provide evidence of the critical role of NaPi-IIa in human renal phosphate handling.

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na/H exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.