I. INTRODUCTION

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In the last decade, a tremendous amount of knowledge has been gathered on the processes by which cells import/export essential ions across their cellular membranes and how individual systems maintain the precise levels of ion concentrations for proper body function. One essential electrolyte is inorganic sulfate, an anion which is required by all organisms for life. Sulfate is required for proper cell growth and development of the organism. It is involved in a variety of important biological processes, including biosynthesis and detoxification via sulfation of many endogenous and exogenous compounds. Sulfate is required for cell matrix synthesis and for the maintenance of cell membranes. Despite its importance in cellular activities, up until 30 years ago it was considered an inert ion, whose precise function was unknown. Only recently have molecules been identified that facilitate cellular sulfate transport to/from the extracellular environment. Such transmembrane movement is of utmost importance, since without it, cells would not be able to regulate the contents of cellular sulfate required for many biological processes, nor would they be able to control sulfate homeostasis in the body. With the use of molecular biological techniques, many families of sulfate transporters, of both prokaryotic and eukaryotic origins, have been cloned in the past 10 years. As expected, sulfate transporters are not only restricted to mammals, but also exist in abundance in a variety of eukaryotic (e.g., birds, fish, amphibians, crustaceans, insects, plants and yeasts) and prokaryotic (i.e., bacteria) species (35, 142, 203, 237). Due to the breadth of this research field, this review focuses solely on the physiology and regulation of mammalian sulfate transporters, a topic which has not been thoroughly reviewed since the cloning of these novel proteins. The aim of this review is to provide a detailed report on the research of membrane sulfate transporters, with the rationale that a tremendous amount of information has been acquired, with over 100 original papers published in this field in the last 10 years.

This review is arranged into sections in chronological order of discovery on topics in sulfate transport physiology. Initially, the physiological role of the sulfate anion and its historical perspective is described in section II. This is followed by a review of early studies that first identified and characterized sulfate transport systems from various mammalian organs, cell lines, and intracellular compartments (sect. III). With the use of molecular and cell biological techniques, the structural identification and functional characterization of cloned sulfate transporters are depicted in section IV. Subsequently, the regulatory factors that control sulfate transporter expression in vivo and in vitro are elucidated in section V, and the pathophysiological conditions associated with defective sulfate transporters are described in section VI. Finally, future research trends are outlined in section VII, describing research areas which have not been extensively investigated, but are likely to be important for studying sulfate transporters in the coming years.

	II. HISTORICAL PERSPECTIVE OF SULFATE

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The sulfate ion in clinical medicine has been regarded as an end metabolite of cysteine and methionine, both sulfur-containing amino acids. Sulfate has been associated with an increase in body acidity and has been shown to lead to a drop in fluid osmolarity of body fluids (91, 222). Despite the fact that sulfate itself does not possess a catalytic function or a role in human energy metabolism, there is outstanding evidence to suggest that sulfate is not a metabolically inert molecule and that it plays a key function in life.

The sulfate ion is the oxidized form of the 16th element of the Periodic Table, sulfur (S⁶⁺) [Latin: sulfur], which is surrounded tetrahedrally by four oxygen molecules (O²) forming the divalent anion SO. In nature, sulfate is an inorganic molecule belonging to the group VI oxyanions, which includes other structurally similar members such as selenate, molybdate, tungstate, and chromate. It is an important anion involved in many physiological processes, having numerous biosynthetic and pharmacological functions. Sulfate is involved in a variety of activation and detoxification processes of many endogenous (including glycosaminoglycans, cerebrosides, steroids, catecholamines) and exogenous (acetaminophen, isoproterenol, ibuprofen, salicylate, -methyldopa) compounds (for extensive reviews, see Refs. 180-182). In the body, sulfate can be directly obtained from the diet (110, 238) or formed by oxidation of sulfur-containing amino acids, cysteine and methionine, present in the diet (132, 209). The degree of contribution by either process toward total sulfate load is yet unknown. Outside the body, sulfate is formed by reaction of the sulfur atom in sulfite (S⁴⁺) with atmospheric oxygen. The sulfite ion (SO) is a precursor to the sulfate ion (SO) and is relatively reactive compared with sulfate. Most of the sulfite in the body is formed from hydrolysis of 3-sulfinylpyruvate by sulfite oxidase found within the intermembraneous space of liver mitochondria (for recent review, see Ref. 44). An early report documented rat liver mitochondria to be able to transport sulfite (48). Furthermore, sulfite was able to weakly cis-inhibit and trans-stimulate sulfate uptake into rabbit renal brush-border membrane vesicles (221), suggesting that it may not be transported by the same system as for the sulfate ion.

Body sulfate homeostasis was suggested to be in part maintained through renal clearance mechanisms (10, 15). The plasma SO levels in humans are generally maintained fairly constantly (±10%) over a 24-h period (165). However, after oral SO loading (i.e., high protein diets), plasma SO can increase up to twice normal levels, with excess SO quickly excreted within 12 h. In contrast, upon fasting, the majority of filtered SO is reabsorbed (165, 185). Renal handling of SO is altered in patients with chronic kidney disorders, there being a marked reduction in tubular SO reabsorption, that is sufficient to prevent an increase in plasma SO (170). At decreased serum sulfate concentrations, decreases in renal sulfate clearance are observed (144). Since sulfate is not extensively bound to serum proteins and the majority of filtered sulfate is reabsorbed, it has been suggested that sulfate is not secreted to any significant extent (15). This renal tubular-mediated process has been suggested to be the mechanism of sulfate homeostasis and is discussed in greater detail in sections III and IV.

One physiological importance of the sulfate anion is in the process of sulfate conjugation of various compounds. For conjugation, sulfate must be activated to form adenosine 3'-phosphate 5'-sulfatophosphate (PAPS), the "universal" sulfate donor. Various sulfotransferase enzymes catalyze the transfer of sulfate from PAPS to various compounds, and these enzymes are located in virtually all tissues of the body. Sulfation (also known as sulfonation) is an important step for the biotransformation and detoxification of xenobiotics (including analgesics, anti-inflammatory agents, and adrenergic stimulants/blockers), steroids, catecholamines, and bile acids (180-182). For a schematic view of the processes involved in the formation, activation, and conjugation of sulfate, see Figure 1. In addition to having a role in the detoxification of numerous toxic substrates (e.g.. phenols, drugs, heavy metals), sulfate conjugation also serves a role in the biosynthetic pathway for the production of numerous biologically active substrates (including steroids, neurotransmitters, bile acids) (182). Sulfate is also essential for the biosynthesis of numerous structural components of membranes and tissues; it is involved in the formation of sulfated glycosaminoglycans (sGAG), major components of cartilage and other tissues, and in the formation of cerebroside sulfate, a constituent in the myelin membranes of the brain (55, 58). The degree of sulfation of various endogenous compounds can affect their physiological activities: increased sulfation of heparan sulfate and dermatan sulfate enhances their anticoagulant activities (19, 188). Furthermore, the importance of sulfation in growth and development has been demonstrated by the increased serum sulfate concentrations in the fetus, pregnant women, and children compared with adults (41, 43, 176, 244). Since sulfate conjugation is an extremely broad research area, with no direct links established to date to sulfate transporters per se, it is not covered in this review. For an excellent recent series of reviews on sulfation, see References 72, 83, 123, 265.

View larger version (21K): [in this window] [in a new window]   Fig. 1. General processes involved in the formation, activation, and conjugation of sulfate. 1, ATP-sulfurylase; 2, APS kinase; 3, sulfotransferases; 4, sulfatases. APS, adenosine 5'-phosphosulfate; PAPS, adenosine 3'-phosphate 5'-sulfatophosphate. [Adapted from Mulder (181).]

Sulfate transport has been extensively studied both at the biomembrane and tissue levels, particularly in intestinal absorption, renal tubular reabsorption, and transport from the liquor compartment of the central nervous system (CNS) (50). Individual cells can obtain sulfate by three distinct mechanisms: 1) intracellular hydrolysis of sulfoconjugates, 2) oxidation of reduced organic sulfur, or 3) transport of sulfate from extracellular fluids into cells. The quantitative contribution of each process to total cellular sulfate levels is not precisely known, but it is believed that without the latter (membrane transport) step, cells would not be able to maintain a sufficiently high enough level of sulfate for all the cellular processes to function properly. It is this latter step that forms the focus of this review.

	III. SULFATE TRANSPORT SYSTEMS

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Since sulfate is a highly dissociated, divalent hydrophilic anion, it cannot pass freely through the phospholipid bilayer of plasma membranes and is therefore dependent on a transport system to allow movement into/out of active cells and cell organelles (such as mitochondria). Transport mechanisms in mammals are also needed for sulfate absorption from the gastrointestinal tract, reabsorption by the renal tubules, and excretion from the cerebrospinal fluid. The notion that sulfate transport through plasma membranes is mediated by a specific protein embedded in the lipid bilayer was proposed about 35 years ago (62). However, only in the last 10 years has structural information been available for proteins that are responsible for these transport mechanisms (see sect. IV). Since intracellular sulfate-containing nucleotides, such as adenosine 5'-phosphosulfate (APS) and PAPS, and the enzymes needed for sulfate activation (sulfotransferases) are present in all animal tissues and cells, all cells would be expected to be equipped with systems for the uptake of sulfate. Before the cloning of specific sulfate transporters, numerous approaches were used to determine and characterize sulfate transport systems in various tissues. These studies are documented below.

The most widely studied organ in terms of sulfate transport, as with many other ion transport systems, has been the mammalian kidney. Early studies in dogs demonstrated that sulfate was freely filtered and extensively reabsorbed by the kidneys (84). Porous membranes such as the renal glomerular basement membrane were shown to freely filter sulfate (242). Stop-flow experiments proposed that the kidney proximal tubule was the major site of active sulfate reabsorption (98). With the use of turbidimetric analysis (16), serum sulfate concentrations in humans were measured to be 270 ± 20 µM (133). In dogs, normal plasma sulfate levels were measured to be 1-2 mM, with fractional excretion being ~10% (84); thus the majority of sulfate was reabsorbed by the kidneys. Tubular transport capacity for sulfate was calculated as ~120 µmol/100 ml filtrate in the dog (149). The active sulfate reabsorption process was shown to be capacity limited and saturable (144). Under physiological conditions, tubular SO reabsorption works near the maximal rate (179), whereas if plasma SO increases, the filtered load of SO quickly exceeds the maximal tubular reabsorption and SO is excreted in the urine. Renal clearance of sulfate increases with increasing serum sulfate concentrations reaching approximately the glomerular filtration rate (GFR). At physiological serum sulfate concentration of 700-1,000 µM in the rat (133, 181), sulfate renal clearance was measured to be ~30% of the GFR.

In proximal tubular cells, sulfate transport systems have been extensively characterized on the luminal brush-border membrane (BBM) and the contraluminal basolateral membrane (BLM), by microperfusion studies in vivo (54, 252-258), with isolated tubules (24) and membrane vesicle studies (85, 150, 152, 197, 221, 251). The principle pathway for secondary active sulfate uptake is initiated across the BBM via sodium-coupled sulfate transport (Na⁺-SO cotransport), driven by the luminal membrane Na⁺ gradient (8, 152, 221, 251). The influx of sulfate into the proximal tubular cell generates an outward sulfate gradient. Early studies demonstrated that exit of sulfate across the BLM occurs via an SO/HCO exchanger (24, 150, 197), which completes the process of transcellular sulfate reabsorption in the proximal tubule. The direct consequence of this process would be to drive HCO uptake into the proximal tubular cell across the BLM, thereby contributing to intracellular pH regulation (i.e., raising the pH). The same scenario would occur across the BBM: backflux of sulfate from the proximal tubular cell to the tubular lumen via a SO/HCO exchanger (117, 196, 245) for HCO, would also drive HCO into the cell, thereby raising intracellular pH. The gain of intracellular HCO could be counteracted by the exit of HCO from the cell by oxalate/HCO exchanger involved in bidirectional transport of oxalate and HCO on both the luminal and basolateral membranes of the proximal tubule (264).

Early uptake studies using radiotracer [³⁵S]sulfate in BBM vesicles (BBMV) demonstrated the presence of a Na⁺-dependent sulfate transport system (221, 251, 253). In rat renal BBMV, the affinity (K_m) for this transport system was estimated to be 600 µM for sulfate and 36 mM for sodium, with a Hill coefficient (n) of ~1.6 (221). This transport system was kinetically characterized to be transporting two Na⁺ with one SO, a stoichiometry consistent with an electroneutral transport mechanism (221, 251). This electroneutral kinetic model was favored for many years, right up until the molecular isolation of this protein structure in 1993 (157), when a more detailed functional characterization demonstrated this protein to be electrogenic (28). Transport characterization of the cloned renal BBM Na⁺-SO cotransporter (NaSi-1; Ref. 157) by electrophysiological recordings (28) demonstrated a kinetic model favoring the transport of three Na⁺ for one SO (Fig. 2, see also sect. IVA1). With regard to substrate specificity, the BBMV Na⁺-SO cotransport activity was shown to be inhibited by thiosulfate, molybdate, and other oxyanions (including chromate and selenate), but not by phosphate or tungstate (253), suggesting that this transport system was unable to mediate phosphate transport. Na⁺-SO cotransport was well characterized in BBMV from rat (8, 152) and rabbit (1, 221, 251) kidney cortex. The presence of a Na⁺-dependent SO transport system was more recently demonstrated by injecting rabbit kidney cortex mRNA into Xenopus laevis oocytes (266) (see also sect. IVA1).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Model of a renal proximal tubular cell with sulfate transport systems. Anions (A) include sulfate, thiosulfate, oxalate, selenate, molybdate, chromate, hydroxyl, or bicarbonate.

In addition to the Na⁺-SO cotransport system present on the BBM, there has been evidence to suggest that sulfate can be transported by an anion exchange (AE) mechanism across the luminal membrane in BBMVs from rat (196), bovine (245), and rabbit (117) kidney cortex. This transport system was shown to mediate an electroneutral anion exchange, with little or no interaction with Cl and to be able to transport bicarbonate, oxalate, acetate, lactate, succinate, and p-aminohippurate (PAH), in addition to sulfate (117). The overall importance and contribution of this transport activity to sulfate reabsorption in proximal tubular cells is yet unknown, nor has this transport system been identified at the molecular level.

The proximal tubular BLM possesses an anion exchange sulfate transport system that transports sulfate out of the cell into the systemic circulation and exchanges sulfate with thiosulfate, hydroxyl ions, bicarbonate, oxalate, and a variety of organic ions (24, 150, 197). In rat renal BLM vesicles (BLMV), the sulfate anion exchanger shows specificity for the exchange of bicarbonate, hydroxyl, thiosulfate, and sulfate ions, but not chloride, phosphate, lactate, or PAH (88, 197). Furthermore, stilbene derivatives DIDS and SITS, characterized as inhibitors of anion exchangers (3, 8, 18, 24, 54, 197) (among other proteins), could inhibit sulfate uptake into BLMVs (8, 85, 88). This BLM transport system has been shown to have different transport kinetics and a different sensitivity to inhibitors than the BBM sulfate anion exchanger (54), suggesting that they are not encoded by the same protein. For example, the apparent inhibitory constant (K_i) for DIDS, probenecid, phenol red, and oxalate (among others) on sulfate uptake is over threefold higher for the BBM than for the BLM sulfate anion exchanger, suggesting that the BLM transporter is more sensitive to inhibition by these compounds than the BBM sulfate transporter (54). Furthermore, upon the cloning of the BLM sulfate anion exchanger (sat-1; Ref. 18), antibodies raised against this protein showed immunocytological staining only on the BLM (118), suggesting that this protein is structurally (in addition to functionally) different to the BBM sulfate anion exchanger (see sect. IVB1).

Despite the identification of several different SO transport systems in renal proximal tubules, which of these plays the predominant role in sulfate homeostasis is still unclear. Since the active reabsorption process is dependent on a normal Na⁺-K⁺-ATPase activity in the proximal tubular cells (183), it has been suggested that a sodium gradient may be important in sulfate uptake (as compared with anion exchange) and that the Na⁺-sulfate cotransporter on the BBM may play the predominant role (17). In the lower urinary tract (i.e., human ureteral epithelial cells), there is evidence of a DIDS-sensitive sulfate/chloride anion exchanger, but not a Na⁺-dependent sulfate transporter (69), suggesting that active sulfate uptake may not be occurring in the lower urinary tract, but is restricted to the renal proximal tubule. For a model of the sulfate transport systems present in the renal proximal tubule, see Figure 2.

The majority of sulfate absorption occurs in the latter part of the small intestine (ileum and jejunum) (183). Sulfate uptake has been studied mostly using membrane vesicles purified from mammalian small intestines. In pig jejunum BBMVs, there is evidence of sulfate transport by both a Na⁺-dependent SO cotransporter and a Na⁺-independent SO anion exchanger (269). Na⁺-dependent sulfate uptake was also demonstrated in BBMV isolated from rabbit (1) and rat (153) ileum, with similar transport properties to the renal BBM transport system. BBMV [³⁵S]sulfate uptake experiments measured the ileal Na⁺-sulfate cotransporter to have a K_m of 520 µM for sulfate in the presence of 100 mM sodium (1), which closely agreed with the sulfate affinity (K_m of 600 µM) of the renal BBMV Na⁺-sulfate cotransporter (221). The proposed kinetic model for this transporter was also suggested to be electroneutral, with two Na⁺ being transported with one sulfate ion (1). As with the renal BBMV Na⁺-sulfate cotransporter, this model was later proven to be incorrect (see sect. IVA1). In BBMV from rabbit ileum, there is evidence of a SO/OH exchange system, which is stimulated by a pH gradient and inhibited by DIDS and SITS (223). A carrier-mediated Cl/SO exchange system has been described in BLMV of the rabbit ileum (224) and rat jejunum (88), with characteristics similar to the renal BLM anion exchanger. The evidence gathered from membrane vesicle studies suggests that the mechanisms of sulfate transport in the small intestinal epithelia are identical to those in kidney proximal tubules. Structural isolation of the small intestinal sulfate transporter has demonstrated this indeed to be the case, with the rat ileal BBM Na⁺-SO cotransporter (195; see sect. IVA1) being identical to the renal BBM Na⁺-SO cotransporter (157).

Carrier-mediated hepatic sulfate uptake was measured in isolated perfused rat liver (22) and isolated hepatocytes (260). Sulfate uptake in hepatocytes was shown to occur by Na⁺-dependent and Na⁺-independent systems, with K_m values for sulfate of 2.3 and 33 mM and maximum velocity (V_max) values of 2.1 and 10 nmol·mg protein¹·min¹, respectively (260). Analysis of the Na⁺ dependency for the Na⁺-sulfate cotransport system indicated an n value of 1.05, suggesting an equimolar stoichiometry for sodium and sulfate transport. Bicarbonate was shown to affect sulfate transport, with sulfate uptake increased by intracellular bicarbonate and competitively inhibited by extracellular bicarbonate (260). Sulfate transport in hepatocytes was further characterized in rat liver basolateral (sinusoidal) plasma membrane vesicles and was demonstrated to be mediated by an anion exchange pathway, through countertransport with intracellular hydroxyl ions (109). This sulfate uptake was saturable with increasing sulfate concentrations (K_m = 16.1 ± 3.9 mM), was stimulated by a pH gradient (pH 8.0 inside, pH 6.0 outside), and was inhibited by probenecid, DIDS, and nigericin (109). Subsequently, a sulfate/HCO anion exchange system was also identified in apical (canalicular) rat liver plasma membrane vesicles, which was stimulated by a bicarbonate gradient (50 mM in, 5 mM out, pH 8.0 in and out) producing a saturable sulfate transport, which could be inhibited by probenecid, acetazolamide, furosemide, DIDS, and SITS (166). Thus several sulfate transport systems exist in hepatocytes, for which the functional roles (i.e., possibly bile synthesis) need to be determined.

There is evidence of an anion exchange DIDS/SITS-inhibitable sulfate transport system present on apical membrane vesicles of bovine tracheal epithelium (64). This sulfate transport system appears to be cis-inhibited and trans-stimulated by chloride, bicarbonate, thiosulfate, selenate, and molybdate, but not by phosphate or arsenate (64). The affinity of the transport system for sulfate was observed to be highest in low ionic strength media (K_m = 130 µM) and decreased in the presence of gluconate (K_m = 680 µM). In agreement, bronchial epithelial cells were shown to transport sulfate by an anion exchanger that was inhibited by external chloride, DIDS, and SITS and was not stimulated by sodium (171).

In human lung fibroblasts, sulfate transport was suggested to occur via several pathways: 1) a high-affinity SITS-sensitive pathway, 2) a low-affinity SITS-sensitive pathway, and 3) a SITS-insensitive pathway (68). At extracellular sulfate concentrations of <100 µM, the predominant pathway was the high-affinity SITS-sensitive component with a K_m of 34 ± 14 µM for sulfate. Under normal serum sulfate (100-500 µM) concentrations, the predominant pathway for sulfate influx was the SITS-sensitive, low-affinity pathway with a K_m of 1 mM for sulfate (68). The SITS-insensitive pathway, for which no K_m for sulfate transport could be determined, was detectable (to a lesser extent) only under serum sulfate concentrations. Arsenate and orthophosphate had no effect on sulfate influx, whereas bicarbonate, molybdate, vanadate, and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux (68). Extracellular chloride was able to inhibit the influx of sulfate and stimulate the efflux of sulfate, suggesting that sulfate is transported across bronchial epithelial cells via an anion exchanger that exchanges chloride for sulfate (67, 68, 171).

Sulfate transport has been extensively studied in the erythrocyte, where it occurs via an anion exchanger known as band 3 (124). Band 3 (130) constitutes the major integral membrane protein of the red blood cell (RBC) (124). Since erythrocytes have a high capacity for rapid exchange of anions, SO exchange has been postulated to share a common transport mechanism with Cl and HCO, a process which is electroneutral in RBCs (2, 3, 32). The primary function of the erythroid band 3 sulfate anion exchanger is to increase the capacity for plasma CO₂ transport by exchanging HCO generated by intracellular carbonic anhydrase for extracellular Cl (125). The physiological roles of the erythroid band 3 protein is discussed in greater detail in section IVB4.

The sulfate anion exchanger band 3 (or AE1; see sect. IVB4) was found to be expressed ubiquitously in all cells studied, including isolated neurons and neurons of the CNS (120). As in the erythrocyte, the function of band 3 in neurons has been postulated to be important for the exchange of HCO with Cl and sulfate (120). Despite this, very little information is available to date on the requirement for sulfate transport systems in neurons. Sulfation plays an important role in the CNS for the biosynthesis of sulfated proteoglycans that are involved in modulating cell interactions in developing nervous tissues (61). Neuronal heparan sulfate proteoglycans are involved in cell adhesion, neural crest migration, and neurite extension (60, 187), whereas chondroitin sulfate proteoglycans (of astrocyte origin) have been shown to inhibit neurite growth (86). Several findings have proposed that the brain may possess a sulfate transport system. Ventriculocisternal perfusion studies have demonstrated that a carrier mechanism for sulfate transport exists in the brain, capable of mediating sulfate transport from the cerebrospinal fluid (CSF) to plasma (50, 147). This proposed mechanism of sulfate transport was shown to be saturable, with the addition of unlabeled sulfate to the perfusate leading to significantly reduced [³⁵S]sulfate clearance. In addition, sulfate transport was markedly reduced by the addition of thiosulfate, a sulfate analog that competed for the same transport mechanism. Since sulfate transport in the brain is important for maintaining CSF sulfate concentration (50, 147), it is likely that similar transport processes may also be required in the brain, where the import of sulfate into cells of the CNS would be needed. Recently, a rat brain sulfate transporter, which is sensitive to DIDS and oxalate, has been characterized in this laboratory (135), confirming that neuronal and/or glial cells contain a functional sulfate transporter (see sect. IVB1).

Sulfate is an essential metabolite utilized by the developing fetus in the synthesis of sulfate mucopolysaccharides, proteins, and steroids (41). In the human placenta, SO transport has been shown to be mediated by a DIDS-sensitive anion-exchange transport system (45). Human placental tissue slices showed concentrative sulfate uptake only in the absence of sodium and at low pH (45). BBMVs isolated from human placenta were shown to contain a sulfate transporter with broad specificities for other oxyanions including selenate, tungstate, molybdate, and chromate (57). Vesicles isolated from human placental trophoblasts expressed both apically and basolaterally located SO transport systems, both being electroneutral with an approximate K_m of 2.5 mM for sulfate and DIDS sensitive (K_i for DIDS = 10 µM) (29). In placental tissue slices and placental BBMV, sulfate uptake was inhibited by salicylate, suggesting salicylate ingestion could compromise fetoplacental sulfate homeostasis (233). To date, there is no evidence of a Na⁺-dependent sulfate transport system in the human placenta. Sulfate transport properties in human placenta are in keeping with an anion exchange system, including the lack of stimulation by sodium, trans-stimulation by bicarbonate, and inhibition by SITS and DIDS (20, 21, 40, 57, 231), suggesting that a sulfate anion exchanger is most likely responsible for sulfate accumulation by the human fetus.

In lactating rat mammary glands, a DIDS-sensitive SO anion exchange system was detected, which was trans-stimulated by chloride, iodide, and sulfate anions (230). Furthermore, selenate and other divalent anions (including molybdate and thiosulfate) were also able to trans-stimulate the efflux of radiolabeled sulfate from lactating rat mammary tissue slices (232). The effect of selenate on sulfate efflux was saturable with an apparent K_m of 270 µM (232). From this study, it was concluded that sulfate and selenate may share a pathway for transport in the lactating rat mammary gland (232). This sulfate transport system may be important for the metabolism of SO by the mammary gland and may help in determining milk anion concentrations.

Anion-exchange carrier-mediated sulfate-transport systems, similar to band 3, have been characterized in a variety of mammalian cell lines, including Ehrlich ascites tumor cells (141), Vero cells (189), human lung fibroblasts (68), Chinese hamster ovary (CHO) cells (65), and skin fibroblasts (66). All of these sulfate transport systems were shown to be mediated by an electroneutral anion exchange system and exhibited high affinity for chloride, bicarbonate, and sulfate. A recent study documented the presence of a sulfate anion exchange (AE) activity in Madin-Darby canine kidney (MDCK) cells (7), a renal epithelial cell line derived from the dog kidney tubule. Functional AE activity was demonstrated in MDCK cells by increased uptake of sulfate at pH 6.0 over pH 7.0 and the inhibition by DIDS and nonhalide anions including molybdate, oxalate, pyridoxal 5'-phosphate, but not phosphate (7). In Ehrlich ascites cells, sulfate flux was shown to be saturable, with a K_m ~2 mM for sulfate, in the absence of chloride (141). To date, there has only been one report of a Na⁺-coupled sulfate transporter in an established mammalian cell line, the OK-E cells, a clonal subline of the proximal tubular opossum kidney (OK) cells (250). Transport kinetics for sulfate interaction were determined: K_m= 2.4 ± 0.2 mM and V_max= 125 ± 15 pmol·mg protein¹·min¹ for sulfate. Hill analysis demonstrated a Na⁺ dependence of sulfate transport with the following values: n = 1.5 and Michaelis contstant K_Na = 23 mM (250). This transport mechanism was stimulated by an acid pH (lowering the pH from 7.4 to 6.4) but was inhibited by DIDS (IC₅₀ for DIDS = 0.9 µM), bicarbonate (IC₅₀ for HCO = 7 mM), sulfite, thiosulfate, chromate, picrylsulfanoate, and ethancrynate (but not phosphate or amiloride). In the absence of extracellular chloride, the sulfate affinity for the carrier (K_m = 500 µM) was increased without affecting its V_max. A similar transport activity was observed in mouse renal BBMV, although the sensitivity to DIDS (IC₅₀ for DIDS ~500 µM) and HCO (~50 mM) was significantly lower than in OK-E cells (250). The sulfate transport system characterized in OK-E cells (250) is quite unique in its mechanism, since it is both a Na⁺-coupled system, with a slightly lower affinity (for sulfate) than the renal proximal tubular BBM Na⁺-SO cotransporter and is extremely DIDS sensitive, a more common feature for sulfate anion exchangers, rather than Na⁺-SO cotransporters which are insensitive to DIDS. Such an interesting (transport kinetic) system has not been observed in an in vivo system.

Mitochondria from rat kidney cortex and liver cells take up sulfate ions from their environment. This transport system was first described as a sulfate/dicarboxylate exchanger, which was also able to transport phosphate (48), suggesting a common transport mechanism for the uptake of sulfate, phosphate, and dicarboxylates into mitochondria. Furthermore, the mitochondrial sulfate transport activity was demonstrated to be mediated by a H⁺ symport carrier (218). The kinetics of sulfate uptake were measured with a calculated K_m value for sulfate of ~300 µM (49). In this study, a competitive relationship was demonstrated between the influxes of sulfate and malonate, whereas sulfate and phosphate showed a mixed mechanism of competition (49). Furthermore, mersalyl and bathophenanthroline showed similar inhibition patterns for sulfate and malonate transport, but not phosphate transport (49), suggesting that sulfate and malonate may bind to the same site whereas phosphate to a different site on the carrier. Early reports showed that parathyroid hormone could stimulate sulfate, phosphate, and arsenate uptake into mitochondria (200), possibly by a common mitochondrial transport system. Since the conversion of thiosulfate to sulfite in mammals is catalyzed by mitochondrial thiosulfate reductase and thiosulfate sulfurtransferase enzymes (129), the physiological function of a mitochondrial sulfate transport system could be important for sulfur metabolism.

More recently, the rat mitochondrial dicarboxylate carrier (DIC) was cloned and showed the ability to exchange sulfate, thiosulfate, and succinate for malate and phosphate uptake in bacterial reconstituted proteoliposomes (76). Unlike the rat DIC, the bovine mitochondrial phosphate carrier (PiC) shows a more narrow substrate specificity, with a preference for high phosphate uptake when phosphate or arsenate is preloaded into liposomes, whereas sulfate and malate had almost no effect on mitochodrial phosphate uptake in reconstituted proteoliposomes (59). It is still unclear whether DIC or PiC encode mammalian mitochondrial sulfate transporters.

Sulfate, being a by-product of the degradation of macromolecules such as glycosaminoglycans, exits lysosomes via a carrier-mediated pH-dependent sulfate transport process (127). This process was shown to be inhibited by DIDS, SITS, phenylglyoxal, 1,2-cyclohexanedione, niflumic acid, and dinitrofluorobenzene, suggesting the lysosomal sulfate transporter has functional similarities with the erythrocyte band 3 anion transporter. However, the potent band 3 inhibitor dipyridamole had no effect on lysosomal sulfate transport, suggesting that there may be some structural differences between the erythrocyte and lysosomal sulfate anion transporters. In an attempt to purify the lysosomal sulfate transporter, a method for reconstitution of transport in artificial membrane vesicles was developed (128). Proteoliposomes were prepared from Percoll density gradient-purified rat liver lysosomes and exhibited saturable sulfate transport with characteristics similar to those in lysosomal membranes, with a K_m value of 155 µM for sulfate, exhibiting a pH dependence and sensitivity to DIDS (128). ATP was shown to markedly stimulate sulfate uptake by rat liver lysosomes that were treated with N-ethylmaleimide, a blocker of the lysosomal proton-translocating ATPase (H⁺-ATPase), with maximal stimulation requiring millimolar concentrations of ATP and neutral pH (36). ATP-stimulated transport exhibited saturation kinetics with a K_m of 175 µM for sulfate, identical to the K_m for lysosomal sulfate uptake at pH 5.0, an ATP-independent process. Exposure of lysosomes to protein kinase A and protein kinase C inhibitors had no effect on the stimulation of sulfate transport by ATP, suggesting that the lysosomal sulfate transport protein does not require protein kinase A or C phosphorylation for expression (36). More recently, using thiol blocking agents, the role of sulfhydryl groups for function of the lysosomal sulfate transport system was examined (37). Monothiol binding reagents p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulfonic acid (p-CMBS), dithiol binding reagents such as CuCl₂, the alkylating agent N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulfate transport (37). NEM exposure led to a sevenfold increase in K_m (867 µM vs. control 126 µM) and a decrease in V_max (99 pmol vs. control 129 pmol·unit -hexosaminidase¹·30 s¹) (37). Similarly, exposure to Cu²⁺ led to an increase in K_m for sulfate to 448 µM and a decrease in V_max to 77 pmol·unit -hexosaminidase¹·30 s¹ (37). These data suggest sulfhydryl groups may play a role in lysosomal sulfate transport through effects on substrate affinity. Despite extensive transport characterization, this protein remains yet to be cloned; however, sulfhydryl binding may appear to be a strategy for the purification of this transporter.

Numerous studies have been aimed at elucidating the mechanisms by which body sulfate homeostasis is regulated. Various pharmacological agents have been shown to modulate serum sulfate levels in vivo. Probenecid, an inhibitor of renal organic anion secretion, increases renal sulfate clearance by ~1.8-fold and reduces serum sulfate levels by ~1.7-fold, without affecting serum levels or renal clearance of uric acid, magnesium, calcium, or phosphorus (53). Administration of a nonsteroidal anti-inflammatory drug (NSAID) salicylic acid (SA) to rats resulted in an approximately twofold increase in renal sulfate clearance (2.13 ± 0.74 vs. 1.09 ± 0.54 ml·min¹·kg¹ in control rats), which led to an approximately twofold decrease in serum sulfate levels (550 ± 120 vs. 1,040 ± 230 µM in controls) (175). SA has been shown to inhibit sulfate transport in the kidney, placenta, and erythrocytes (5). SA administration to rats inhibited sulfate transport in both renal BBMVs and BLMVs: K_i for SA values were calculated at ~20 mM for BBMV Na⁺-sulfate cotransport and 1-2 mM for BLMV sulfate/bicarbonate exchange (52). Due to the lower K_i for SA value measured in BLMVs, the inhibitory effect of SA may be predominantly inhibition of the BLM sulfate transporter, which may (at least in part) be responsible for the SA-induced increases in renal sulfate clearance (52). Chronic aspirin administration to healthy human individuals caused a small but significant decrease in serum sulfate levels (after 8 days of administration), but unlike in rats, no apparent change in renal clearance of sulfate (or creatinine) was observed (172), suggesting chronic SA administration has little effect on sulfate homeostasis in humans.

Chronic administration of two other NSAIDs, naproxen and sulindac, to arthritic patients suffering from renal impairment led to a significant (10-25%) increase in serum sulfate concentration and a significant (20-33%) decrease in renal clearance of sulfate and creatinine, compared with younger subjects with normal renal function (174). Naproxen and sulindac produced no changes in renal clearance or serum levels of other electrolytes, including sodium, phosphate, potassium, magnesium, and calcium (174), suggesting their effects were specific on renal sulfate handling. The effect on renal function by naproxen and sulindac was less pronounced than by chronic SA administration in rats (175) and was postulated to be a consequence of mild renal impairment in older arthritic patients.

Acetaminophen (AA) administration to normal rats decreased serum free sulfate concentrations by sevenfold (from 1,070 ± 130 to 150 ± 10 µM), whereas it had no effect in rats with renal dysfunction or renal failure (143). In normal rats, AA is conjugated with sulfate forming AA sulfate, which is excreted by the kidneys, thereby depleting free sulfate in the body, which leads to the formation of other metabolites (i.e., acetaminophen glucuronide) and AA-induced hepatotoxicity (143). In rats with renal dysfunction/failure, retention of free sulfate occurs, which prevents the depletion of serum sulfate by AA, permitting sufficient sulfate availability for the biotransformation of AA to AA sulfate (143). When serum sulfate levels were raised to ~1.5 mM in rats (by sulfate infusion), AA had no effect on the high renal clearance of sulfate during hypersulfatemia (144). Injection of mice with N-acetylcysteine, a compound which decreases AA-induced toxicity, led to increases in serum sulfate concentrations and prevented the depletion of serum sulfate by AA (101). These studies demonstrate the importance of free sulfate levels in the body on the eliminination of drugs subject to sulfate conjugation and the pronounced serum concentration dependence of renal sulfate clearance on sulfate homeostasis.

	IV. CLONING OF SULFATE TRANSPORTERS: THE MOLECULAR BIOLOGY ERA

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A.  Na⁺-Dependent Sulfate Transporters

1.  NaSi-1

The first functional mammalian sulfate transporter was isolated in 1993 using the Xenopus laevis oocyte expression cloning system (157). Previous attempts to purify plasma membrane sulfate transporters using biochemical techniques were unsuccessful. Up until the early 1990s, sulfate transport systems had only been characterized using biochemical techniques such as membrane vesicular uptake assays as well as perfusion studies using radiolabeled [³⁵S]sulfate uptakes. In the late 1980s, the first plasma membrane transporter to be isolated using the Xenopus oocyte expression cloning system was the intestinal Na⁺-glucose cotransporter, SGLT-1 (97). The Xenopus oocyte expression system, which was pioneered by Gurdon et al. in the early 1970s (87), relies on the microinjection of poly(A)⁺ RNA (purified from any source of tissue) into the cytoplasm of Xenopus oocytes. Proteins are then translated, folded, modified, and sorted to their respective compartments (to the cell membrane for plasma membrane proteins) by the oocytes. The newly synthesized proteins can then be functionally characterized by either radiotracer uptakes, electrophysiological measurements, or receptor binding assays. A cDNA library can then be generated from the poly(A)⁺ RNA with the functional activity, and the same functional assay is then used to isolate the cDNA(s) of interest by expression cloning. For a recent methodological review of this technique, see Reference 162. The beauty of this technique is that it does not rely on sequence information but rather a good functional assay to permit the initial expression and subsequent cloning of a novel protein. This powerful heterologous expression system was applied extensively in the 1990s for the isolation of novel membrane transporters, receptors, and ion channels (for reviews see Refs. 162, 235) and is still being used today in many laboratories, with varying degrees of success.

For the expression of sulfate transporters, the X. laevis oocyte system was first implemented in 1990. Injection of poly(A)⁺ RNA from rabbit renal cortex into Xenopus oocytes led to a three- to fourfold stimulation of Na⁺-dependent SO transport (Na⁺-sulfate cotransport) above controls (water-injected oocytes), assayed by radiotracer [³⁵S]sulfate uptake (266). Size fractionation of this poly(A)⁺ RNA led to a further increase (6- to 7-fold stimulation above controls) in Na⁺-sulfate cotransport, permitting transport kinetic parameters to be determined: sulfate interaction, K_m = 370 ± 50 µM and V_max = 108 pmol sulfate·oocyte¹·h¹; Na⁺ interaction, K_m = 21 ± 2 mM, V_max = 105 ± 5 pmol sulfate·oocyte¹·h¹, and n = 1.6 (266). Similarly, injection of poly(A)⁺ RNA from rat kidney cortex into Xenopus oocytes led to a three- to fourfold stimulation of Na⁺-sulfate cotransport (above controls), which allowed transport kinetics to be determined: sulfate interaction, K_m = 600 ± 100 µM and V_max = 37.3 ± 2.2 pmol sulfate·oocyte¹·h¹; Na⁺ interaction, K_m = 33.8 ± 3.4 mM, V_max = 26.1 ± 2.3 pmol sulfate·oocyte¹·h¹, and n = 2.5 ± 0.5 (155). On the basis of this preliminary data, a rat kidney cortex cDNA library was constructed, and upon screening this library for Na⁺-sulfate uptake in Xenopus oocytes, a cDNA named NaSi-1 (for Na-sulfate inorganic cotransporter-1) was isolated by expression cloning (157). NaSi-1 cRNA injected into Xenopus oocytes led to a time- and dose-dependent stimulation of Na⁺-dependent SO uptake, for which the following transport kinetics were determined: sulfate interaction, K_m = 620 ± 80 µM, V_max = 42.7 pmol sulfate·oocyte¹·min¹; Na interaction, K_m = 16.8 ± 2.9 mM, V_max of 17.0 ± 1.4 pmol sulfate·oocyte¹·h¹, and n = 1.8 ± 0.4 (157). These transport kinetics resemble closely the activity of the BBM Na⁺-SO cotransporter of the rat kidney proximal tubule (152, 184).

NaSi-1 transport kinetics were then further characterized in Xenopus oocytes by electrophysiological recordings using current- and voltage-clamp techniques. Superfusion of NaSi-1-injected oocytes that were current-clamped with 1 mM sulfate led to a 12-mV depolarization of the cell membrane (28). In voltage-clamped NaSi-1-injected oocytes, sulfate induced an inward current that was dependent on both Na⁺ and sulfate concentrations, with a calculated K_m for Na⁺ and sulfate of ~70 mM and 100 µM (28), respectively, suggesting electrophysiological determination of NaSi-1 affinity for sulfate is somewhat higher than by radiotracer uptake studies (157). For Na⁺ interaction, the Hill coefficient was measured electrophysiologically to be 1 and 2.8 for sulfate and Na⁺, respectively (28), suggesting that three sodiums are transported per one sulfate ion. This was the first study to demonstrate that the renal Na⁺-SO cotransporter NaSi-1 was in fact not electroneutral (as previously thought), but rather an electrogenic cotransporter of sulfate and Na⁺, with a stoichiometry of 1:3, with one additional Na⁺ being responsible for a net positive charge entering the cell. Thiosulfate and selenate created similar currents as with sulfate perfused to NaSi-1-expressing oocytes, allowing for the determination of K_m for thiosulfate (85 ± 50 µM) and selenate (580 ± 90 µM) (28). This was the first evidence to suggest that NaSi-1 can in fact transport thiosulfate and selenate, in addition to sulfate. To determine whether the three substrates bind to the same or different regions on the NaSi-1 protein, the compounds were added in combination to NaSi-1-injected oocytes. Sulfate was unable to induce an additive current when added to oocytes perfused with thiosulfate or selenate (28), suggesting that all three substrates were most likely transported on the same site of the NaSi-1 protein. Furthermore, perfusion of NaSi-1-injected oocytes with sulfate at various pH values (pH 6.3, 7.3, and 8.3) did not evoke different currents (28), suggesting NaSi-1 activity is not altered by changes in pH.

The NaSi-1 cDNA contains 2,239 bp [including a poly(A) tail] and encodes a protein of 595 amino acids (66.05 kDa), with the hydropathy profile suggesting a protein with at least eight transmembrane domains (TMDs) (157). In vitro translation of NaSi-1 cRNA in rabbit reticulocyte lysate resulted in a protein of expected size, and in the presence of microsomes, the protein size was slightly increased, suggesting possible glycosylation. Northern blot analysis showed two mRNA transcripts of 2.3 and 2.9 kb in kidney (more abundant in cortex than in papilla/medulla) and small intestinal mucosa of rats. NaSi-1 protein localization was confirmed to the BBM of rat renal proximal tubular cells by immunocytochemistry using a NaSi-1 peptide-specific polyclonal antibody (148). These experiments were the first to structurally characterize a membrane protein encoding a Na⁺-SO cotransport system in renal and small intestinal BBMs.

The lack of endogenous NaSi-1 protein expression in any mammalian renal or intestinal cell lines (Markovich, unpublished data) led to several studies being initiated at expressing NaSi-1 protein in a cell culture model, in addition to the existing Xenopus oocyte expression system. NaSi-1 cDNA was transfected into the MDCK cell line (199) and into the baculovirus-driven Sf9 insect cells (80). In both systems, NaSi-1 protein expression was characterized functionally. NaSi-1 stably expressing MDCK cells led to a fourfold increase in Na⁺-sulfate cotransport (compared with vector only transfected cells), which was predominantly expressed at the apical cell surface (199). This result suggested that the sorting behavior of NaSi-1 to the apical membrane is identical to that observed for the renal BBM proximal tubular Na⁺-sulfate cotransporter in vivo (148, 152, 184). NaSi-1 expressing Sf9 cells demonstrated a 60-fold higher Na⁺-sulfate cotransport activity compared with noninfected cells (80). The calculated K_m for sulfate in NaSi-1 expressing Sf9 cells was 300-400 µM, in close agreement with the values obtained in NaSi-1-expressing Xenopus oocytes (157) and the Na⁺-sulfate cotransport activity in renal BBMVs (152, 184). With the use of a NaSi-1 polyclonal antibody, Western blotting and immunoprecipitation detected NaSi-1 proteins of 55-60 kDa in size in NaSi-1-expressing Sf9 cells (80). Recently, this laboratory has generated OK cell lines stably expressing NaSi-1 (unpublished data), which will be used to study the sorting mechanisms and posttranslational regulation of the NaSi-1 protein. To identify the possible involvement of either (or both) phospholipase C (inositol trisphosphate/diacylglycerol) and adenylate cyclase (cAMP) signaling pathways on NaSi-1 function, this laboratory has recently tested pharmacological activators of protein kinase A (8-bromo-cAMP) and protein kinase C (sn-1,2-dioctanoylglycerol; DOG) on NaSi-1 expression in Xenopus oocytes. Our data showed that both 8-bromo-cAMP and DOG inhibited NaSi-1-induced sulfate transport activity in Xenopus oocytes (unpublished data), suggesting possible involvement of phosphorylation for protein function. For a recent review on the membrane trafficking of mammalian sulfate transporters, see Reference 154.

A) OTHER NASI-1 PROTEINS. Recently, this laboratory cloned two additional mammalian NaSi-1 orthologs: the mouse NaSi-1 (mNaSi-1) cDNA (9) and the human NaSi-1(hNaSi-1) cDNA (136). These cDNAs encode proteins that are very similar both structurally and functionally to the rat NaSi-1 (designated from now as rNaSi-1), with the major differences outlined below. By comparison of the amino acid sequences using the ClustalW alignment program, mNaSi-1 shares 93.6% identity and 96% similarity with rNaSi-1, hNaSi-1 shares 82.9% identity and 88% similarity with rNaSi-1, and mNaSi-1 shares 81% identity and 87% similarity with hNaSi-1 (see Table 1). As with rNaSi-1, hNaSi-1 encodes a protein of 595 amino acids, whereas the mNaSi-1 protein is one amino acid shorter at 594 amino acids, with a glutamate missing at residue 314 of rNaSi-1 and hNaSi-1. All three proteins have a calculated molecular mass of 66.1 kDa, comprising 13 putative TMDs (see Fig. 3), predicted by the TopPred2 program (261). Slight differences are present in the locations of putative consensus sites on the three NaSi-1 homologs. Three putative N-glycosylation sites are present in both rNaSi-1 (at Asn¹⁴⁰, Asn¹⁷⁴ and Asn⁵⁹¹) and mNaSi-1 (Asn¹⁴⁰, Asn¹⁷⁴ and Asn⁵⁹⁰) proteins, whereas hNaSi-1 has four putative N-glycosylation sites (at Asn¹⁴⁰, Asn¹⁷⁴, Asn²⁰⁷, and Asn⁵⁹¹). The significance of these sites on NaSi-1 function is presently being investigated. Preliminary data from this laboratory suggest that Asn¹⁴⁰ and Asn⁵⁹¹ on rNaSi-1 may be true glycosylation sites that are essential for protein function. Mutagenesis of these aspargines to serines led to a total loss of Na⁺-sulfate cotransport activity in Xenopus oocytes (unpublished data), most likely due to defective trafficking of the protein to the plasma membrane, as demonstrated previously for N-glycosylation mutants of the renal Na⁺-phosphate cotransporter NaPi-2 (96). Initially, using the Kyte and Doolitle algorithm, the rNaSi-1 protein was predicted to contain 8 TMDs (157); however, more recently using the TopPred2 program (261), the NaSi-1 proteins were suggested to contain 13 putative TMDs (Fig. 3). This 13-TMD model was confirmed by two other web-based programs, TMPred (103) and Sosui (99). In this model, there is a large intracellular loop between TMDs four and five, which was previously predicted to be an extracellular loop in the eight-TMD model, where two putative N-glycosylation sites (Asn¹⁴⁰ and Asn¹⁷⁴) were present (157). Despite the fact that mutating the putative glycosylation site Asn¹⁴⁰ (to serine) in this large loop on the rNaSi-1 protein led to a loss of sulfate uptake in Xenopus oocytes, this result could be due to an alteration in protein folding/sorting and not due to a loss of glycosylation, as shown previously for the NaPi-2 protein (96). Furthermore, the requirement of the N-glycosylation sites for protein sorting and function needs to be determined by the expression of the mutated proteins in a mammalian renal proximal tubular cell line (e.g., OK cells or LLC-PK₁ cells), to rule out that Xenopus oocytes have a different sorting process when compared with mammalian cells. Furthermore, all three NaSi-1 proteins contain numerous putative phosphorylation sites: 1) one cAMP/protein kinase A site (Thr²⁴⁰ on hNaSi-1, Thr⁴⁰⁴ on mNaSi-1, Thr⁴⁰⁵ on rNaSi-1); 2) several protein kinase C sites (rNaSi-1: Ser²¹³, Thr²¹⁸, Ser²³⁰, Thr³²², and Thr⁴²³; mNaSi-1: Ser²¹³, Thr²¹⁸, and Ser²³⁰; hNaSi-1: Ser⁷⁴, Thr²⁰⁹, Ser²¹³, Thr²³⁰, Thr²³⁶, and Thr⁴²³); 3) one tyrosine kinase site (rNaSi-1 and hNaSi-1: Tyr³⁹ , none present on mNaSi-1); and 4) numerous putative casein kinase II and N-myristoylation sites. The functional significance of all of these putative sites still needs to be determined. Of particular interest is a 17-amino acid-long consensus sequence motif (TSFAFLLPVANPPNAIV) called the "sodium:sulfate symporter family signature" (PROSITE PS01271) situated at amino acids 523 to 539 of rNaSi-1, which is highly conserved among the NaSi-1 proteins, as well as with other structurally related proteins, including SUT-1 (82) and the Na⁺-dicarboxylate cotransporters (190) (see below and Table 1). The significance of this motif is yet unknown.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Secondary structure model of the rNaSi-1 protein. Topology is based on the prediction by the TopPred2 program (261). The NH2 terminus is intracellular, and the COOH terminus is extracellular. Putative consensus sites are indicated.

B) FUNCTIONAL CHARACTERIZATION. Substrate specificities are shared among the three NaSi-1 protein homologs; however, slight alterations are observed in their substrate kinetics for Na⁺ and sulfate interactions (see Table 2). The affinites (K_m) for sulfate are in close agreement for all three NaSi-1 proteins; however, the maximal transport velocity (V_max) for sulfate uptake was shown to be fourfold higher in hNaSi-1 (136) than for either of the rodent NaSi-1 proteins (9, 157). This is intriguing since higher serum SO levels have been measured in rodents (~1 mM in rat and mouse) and nine other mammalian species (including guinea pig, rabbit, cat, sheep, goat, cow, pig, monkey, and horse) than in humans (~300 µM) (10, 16, 133). The lower levels of SO in humans were suggested to be due to slower rates of SO formation rather than to an increased renal clearance of this anion (144). Such characteristics would make humans more susceptible to serum SO depletion by xenobiotic drugs that are metabolized to SO conjugates. Thus the expression of a higher capacity SO transporter (hNaSi-1) in the human kidney is quite advantageous, particularly when the serum SO levels are depleted by high doses of substrates for glutathione conjugation and sulfonation (192), and rapid repletion of SO is needed through tubular reabsorption. In fact, it has been demonstrated that upon severe depletion of endogenous SO, almost complete reabsorption of SO occurs in the human kidney (144). Since humans may be more susceptible to SO depletion (by many forms of exogenous substrates) and the rate-limiting step of renal SO reabsorption has been suggested to be the BBM Na⁺-SO cotransporter (17), it is highly likely that hNaSi-1 plays a key role in total body SO homeostasis in humans.

As with the rodent NaSi-1 proteins, hNaSi-1-induced Na⁺-SO cotransport in Xenopus oocytes was significantly inhibited by (1 mM) thiosulfate, selenate, molybdate, and tungstate (in order of potency), whereas phosphate, oxalate, cholate, probenecid, and DIDS had no significant effect (136). Interestingly, unlike the rodent NaSi-1 proteins, hNaSi-1-induced Na⁺/SO cotransport in Xenopus oocytes was significantly inhibited by (1 mM) citrate and succinate; however, succinate was not transported in NaSi-1-expressing oocytes (136), suggesting that in the absence of sulfate in the media, dicarboxylates may competitively bind for the sulfate binding site on hNaSi-1 but themselves are unable to act as transport substrates. To test the extent of hNaSi-1 involvement in mediating Na⁺-SO cotransport in the human kidney, hybrid depletion was performed using hNaSi-1-specific oligodeoxyribonucleotides (ODNs) annealed to human kidney mRNA. hNaSi-1 antisense ODNs prevented >90% of the induced Na⁺-SO uptake by human kidney mRNA, whereas sense ODNs had no effect (136), suggesting that hNaSi-1 is the major functional Na⁺-SO cotransporter in the human kidney.

A very detailed tissue distribution was determined for both mNaSi-1 and hNaSi-1 mRNA expression. By Northern blotting and RT-PCR analysis, out of 23 different tissues, mNaSi-1 was strongly detected in RNA from kidney, duodenum/jejunum, ileum, and colon, with lower levels observed in cecum, testis, adrenal