Min Hwang Chang

The human tumour suppressor gene SLC5A8 expresses a Na+–monocarboxylate cotransporter

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 Km 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.

Divalent metal-ion transporter DMT1 mediates both H+ -coupled Fe2+ transport and uncoupled fluxes

The H+ -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H+ electrochemical gradient. At low extracellular pH (pHo), H+ binding preceded binding of Fe2+ and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pHo, and at pHo 7.4 we observed Fe2+ transport that was not associated with H+ influx. His272 → Ala substitution uncoupled the Fe2+ and H+ fluxes. At low pHo, H272A mediated H+ uniport that was inhibited by Fe2+. Meanwhile H272A-mediated Fe2+ transport was independent of pHo. Our data indicate (i) that H+ coupling in DMT1 serves to increase affinity for Fe2+ and provide a thermodynamic driving force for Fe2+ transport and (ii) that His-272 is critical in transducing the effects of H+ coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe2+ transport in the absence of a H+ gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H+ -uncoupled facilitative Fe2+ transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).

Renal physiology of SLC26 anion exchangers

Purpose of reviewThe multifunctional anion exchanger family (Slc26) encompasses 11 identified genes, but only 10 encode real proteins (Slc26a10 is a pseudogene). Most of the Slc26 proteins function primarily as anion exchangers, exchanging sulfate, iodide, formate, oxalate, hydroxyl ion, and bicarbonate anions, whereas other Slc26 proteins function as chloride ion channels or anion-gated molecular motors. The aim of this review is to present recent studies on the molecular function of the Slc26 family and its role in renal physiology and pathophysiology. Recent findingsIn proximal tubules, Slc26a1 (Sat-1) mediates sulfate and oxalate transport across the basolateral membrane, while Slc26a6 (CFEX, Pat-1) mediates a variety of anion exchange at the apical membrane to facilitate transcellular sodium chloride absorption. Targeted deletion of murine Slc26a6 leads to intestinal hyperabsorption of oxalate, hyperoxaluria, and kidney stones. Slc26a4 (pendrin) and Slc26a7 are expressed in intercalated cells, and are involved in acid–base homeostasis and blood pressure regulation. Messenger RNA for Slc26a2, Slc26a9, and Slc26a11 is also present in the kidney, yet the roles of these family members in renal physiology or pathophysiology are not clear. SummaryMembers of this multifunctional anion transporter family play evolving roles in the etiology of nephrolithiasis (Slc26a6) and hypertension (Slc26a4 and Slc26a6). Other Slc26 family members (Slc26a2, Slc26a9, Slc26a11) express mRNA in the kidney but their roles in renal physiology are not yet known.

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl−-HCO3− exchanger and Cl− channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl−-HCO3− exchange and Cl− currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl− currents with only a modest affect on nCl−-HCO3− exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.

Synthesis, biological, and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.

Entry to "HCO3 tunnel" revealed by SLC4A4 human mutation and structural model

Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na+ bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge-charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was “repaired” by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO_{3}^{-}}\) \end{document} permeation.

Targeting the proton-coupled folate transporter for selective delivery of 6-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of de novo purine biosynthesis in the chemotherapy of solid tumors.

The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-l-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-l-glutamic acid (compound 3), respectively) were inhibitory, with 2 ≫ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ∼50 and 75%, respectively. By an in situ GARFTase assay, 2 was ∼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors.

Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption

The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5-methyltetrahydrofolate (5-MTHF) due to an increase in K(m) and decrease in V(max) in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K(m) for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K(m) mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.