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).

Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration. freshwater adaptation; immunohistochemistry; sulfate transporter; renal proximal tubule SULFATE IS ESSENTIAL for a variety of metabolic and cellular processes, including production of highly sulfated proteoglycans by chondrocytes, detoxification, and elimination of xenobiotics and endogenous compounds by sulfoconjugation in the liver and kidney, and biosynthesis of sulfated hormones such as gastrin and cholecystokinin (27). Mechanisms regulating the levels of plasma sulfate are therefore essential for the maintenance of normal physiology. In mammals, the regulation of sulfate homeostasis is largely determined by the kidney, with the major fraction of filtered sulfate being reabsorbed in the proximal tubule. Two sulfate transporters have been identified that are involved in this reabsorption of sulfate from the glomerular ultrafiltrate: solute carrier family 13a1 (Slc13a1; NaSi-1) and solute carrier family 26a1 (Slc26a1; Sat-1). Slc13a1 is an electrogenic Na ϩ -dependent sulfate transporter (alternatively called Na ϩ -SO 4 2Ϫ cotransporter) that is located in the apical membrane of renal proximal tubule cells and mediates entry of Na ϩ -SO 4 2Ϫ with a stoichiometry of 3:1 (5, 24). Slc26a1 is sulfate/anion exchanger mediating SO 4 2Ϫ efflux across the basolateral membrane in exchange for HCO 3 Ϫ (17). Physiological significance of this regulatory system is well established in mammals through targeted disruption of the Slc13a1 gene (9).Physiological studies on sulfate homeostasis also have been conducted in nonmammalian systems, including those of birds (12,40,44,45), bivalve (11), and fish (10, 36, 37, 43, 46 -49). However, li...

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO3 precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO 3 Ϫ secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na ϩ /HCO 3 Ϫ cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li ϩ /nHCO 3 Ϫ cotransport; HCO 3 Ϫ independent, DIDS-insensitive transport; and increased basal intracellular Na ϩ accumulation. fNBCe1 is a voltage-dependent Na ϩ /nHCO 3 Ϫ cotransporter that rectifies, independently from the extracellular Na ϩ or HCO 3 Ϫ concentration, around Ϫ60 mV. Na ϩ removal (0Na ϩ prepulse) is necessary to produce the true HCO 3 Ϫ -elicited current. HCO 3 Ϫ addition results in huge outward currents with quick current decay. Kinetic analysis of HCO 3 Ϫ currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher Km) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential ϭ Ϫ80 mV; [HCO 3 Ϫ ] ϭ 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO 3 Ϫ secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence. mefugu; euryhaline teleost; sodium-bicarbonate cotransporter; bicarbonate secretion; acid-base; Xenopus oocyte; electrophysiology; intracellular pH; membrane current MARINE FISH LIVE IN A HYPEROSMOTIC environment, yet ionic strength of their blood plasma is similar to that of land animals. Thus these fish are in constant danger of dehydration by the inevitable osmotic loss of water through the gill. To restore body water, they drink seawater (28). However, large quantities of salts are ingested with seawater (ϳ450 mM NaCl). This excess salt must be eliminated to maintain blood osmolarity (ϳ350 mosM). The teleost kidney cannot produce urine more concentrated than the blood plasma (28). Therefore, the ingested seawater is partially desalted in the esophagus, which absorbs Na ϩ and Cl Ϫ by active and passive transport pathways (21, 24), before it enters the stomach. The desalination of the ingested seawater continues at the foregut by active transport of monovalent ions from the lumen into the blood. The desalting process reduces the osmolarity of gut fluid and allows passive absorption of water across the hindgut epithelium. The excess monovalent ions (mainly Na ϩ and Cl Ϫ ) are eliminated by active salt tra...

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Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO3− to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO3− from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO3− excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na+/bicarbonate cotransporter) and apical Slc26a6 (Cl−/HCO3− exchangers).

Boron is a vital micronutrient and is toxic at high concentrations, however, little is known about whole‐body boric acid homeostasis in animals. Slc4a11 was reported to function as a Na+‐coupled borate transporter in mammals, presumably for borate absorption. However, seawater (SW) contains 0.4 mM boric acid, and the bladder urine of a euryhaline pufferfish mefugu (Takifugu obscures) in SW contains ~20 mM boric acid. Therefore, mefugu kidney is a good model to study a borate efflux system. In the mefugu kidney, a paralog of Slc4a11 (Slc4a11A) was markedly induced after transfer to SW and localized to the apical membrane of renal tubules. When Xenopus oocytes expressing Slc4a11A were voltage‐clamped at a holding potential of −60 mV and exposed medium containing borate, intracellular pH was increased and an outward current (anion influx) was observed. The borate current was not altered when Na+ was replaced with other cations such as choline and Li+, but was eliminated when Na+ was replaced with borate chelating agent NMDG. The borate (boron) influx was confirmed by elemental analysis of the oocytes. These results indicate that Slc4a11A is a Na+‐independent B(OH) 4− channel which is suitable for borate secretion, and clarify at first the mechanism of the borate efflux system in animal that prevent marine fishes from the toxic effects of borate.

Euryhaline pufferfish (Takifugu obscurus, mefugu) can live in freshwater (FW) with ion concentration less than 1/100 of its body fluid or acclimate to seawater (SW) which has more than 3 times the ion concentration of its body fluid. However, the body‐fluid ion concentrations are maintained stable by the function of epithelial cells in the gill, intestine, and kidney which transport ions directionally and selectively. To understand the mechanisms of the fish body‐fluid homeostasis at the molecular level, there are several essentials: identification of transporters expressed in the epithelia, determination of transporter localization, and characterization of transporter activities. Analyses of various transporter mRNA levels in mefugu tissues identified increases for Slc26a6A and Slc26a6B in the intestine and the kidney of SW‐acclimated mefugu. To study their functions for SW acclimation, we subcloned the cDNAs into a Xenopus expression vector and assayed their functions using microelectrodes in oocytes.The electrophysiological analyses of the oocytes revealed that (a) Slc26a6A and Slc26a6B are electrogenic Cl−/nHCO3− exchangers; (b) Slc26a6A and Slc26a6B are also exchangers of Cl−/formate, Cl−/oxalate, and Cl−/SO42−; and (c) the slope of I–V relationship of Slc26a6A in the presence of SO42− is 70 times higher than that of Slc26a6B. These results suggest significant roles for Slc26a6A and Slc26a6B in intestinal HCO3− secretion and a role for Slc26a6A in renal SO42− excretion during SW acclimation.This work was supported by MEXT Japan, NIH (EY017732) and CFF grant (Romero‐06G0).