1Departments of Medicine (Nephrology Unit) and 2Pharmacology & Physiology, University of Rochester Medical Center, Rochester, New York; 3Molecular and Vascular Medicine Unit and 4Renal Division, Beth Israel Deaconess Medical Center, Boston; 5Department of Medicine, Harvard Medical School, Boston, Massachusetts
Submitted 4 February 2005 ; accepted in final form 30 March 2005
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ABSTRACT |
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AE1/SLC4A1 is the principal integral membrane protein of the erythrocyte. In concert with carbonic anhydrase, it participates in CO2 metabolism and pulmonary gas exchange (56). AE1 is also expressed in the basolateral membrane of the acid-secreting intercalated cell of the kidney collecting duct. AE1 mutations are associated with spherocytic anemia and distal renal tubular acidosis (1). AE1 also plays an important role in mitotic cytokinesis during the proliferative steps of late erythroid development (45). AE2/SLC4A2 is found in many tissues and, unlike AE1, is inhibited by protons in the physiological range and activated by hypertonicity, suggesting a role in intracellular pH (pHi) homeostasis and cell volume regulation (1921, 23, 60). Absence of AE2/SLC4A2 in the mouse leads to gastric abnormalities and achlorhydria, with death occurring at or before weaning (13). Human mutations of the electrogenic Na+-HCO3 cotransporter (NBC) isoform 1 (NBCe1)/SLC4A4 are associated with severe proximal renal tubular acidosis with ocular abnormalities (22). The electroneutral Na+-HCO3 cotransporter 1 (NBCn1)/SLC4A7-knockout mouse exhibits blindness and auditory impairment (5). RNA interference (RNAi) gene knockdown of the borate transporter 1 (BTR1)-electrogenic Na+-coupled borate transporter 1 (NaBC1)-SLC4A11 in cultured human cells inhibits [3H]thymidine uptake in a manner that can be rescued by borate supplementation (43).
The SLC26 anion transporter family encodes polypeptide products of at least 10 genes (for review, see Ref. 37). The original mammalian member of this family, the sulfate anion transporter (SAT-1/SLC26A1), was expression cloned as a sulfate transporter (4). Additional family members were identified by positional cloning of disease loci for diastrophic dysplasia sulfate transporter (DTDST/SLC26A2) (17), congenital Cl-losing diarrhea [downregulated in adenoma (DRA)/SLC26A3] (18, 54), and Pendred syndrome (pendrin/SLC26A4) (12), as well as by genomic homology searches (32). The SLC26 AEs exhibit a wide range of anion selectivity. DTDST/SLC26A2 appears to mediate HCO3-inhibitable sulfate transport (51). Pendrin/SLC26A4 exchanges I or Cl for Cl or HCO3 but can also transport formate. SLC26A3 is primarily a Cl/HCO3 exchanger, whereas SLC26A6 transports a broad range of anions, including oxalate (26, 65). Their mechanisms of transport remain controversial (7, 27), and prestin/SLC26A5 may move bound intracellular anion through a portion of the transmembrane electrical potential without completing anion translocation and transrelease (42).
The apically localized SLC26 polypeptides SLC26A3/DRA, SLC26A4/pendrin, and SLC26A6 are upregulated by coexpressed CFTR (7, 8, 27). SLC26A3 conversely can upregulate CFTR channel activity through binding of its sulfate transporter and anti--factor antagonist (STAS) domain to the R domain of CFTR (28). SLC26A3 is also thought to interact with CFTR (28) and with Na+/H+ exchanger 3 (NHE3) (31) indirectly through common interactions with postsynaptic density-95/Drosophila disk large/zonula occludens-1 homology (PDZ) domain proteins such as NHE regulatory factor (NHERF)/NHE3 kinase A regulatory protein (E3KARP).
We have previously cloned and characterized the entire family of nematode NHEs nhx-1nhx-9 (39) and have shown that loss of the intestine-specific nhx-2 gene product leads to a starvation phenotype and increases longevity (38). Toward the goal of establishing C. elegans as an experimental system for the study of pH regulation in an intact animal, we have initiated characterization of putative C. elegans anion transporters homologous to mammalian Cl/HCO3 exchangers. Herein we present the cDNA sequences, cellular and subcellular localization, and initial functional studies of these novel proteins.
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METHODS |
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Promoter transgene analysis. As described previously (39), transcriptional promoter fusions to green fluorescent protein (GFP) were created by performing PCR amplification of a 4-kb 5' upstream genomic sequence for each isoform and using restriction site-tagged oligonucleotide primers with a mutant start codon complement (ATG to TTG). These PCR products were cloned into the complementary sites of the five synthetic introns-containing vector pFH6.II (F. Hagen, University of Rochester, Rochester NY) derived from pPD95.81 (A. Fire, Carnegie Institution of Washington, Baltimore, MD) to create the four SLC4-related plasmids pFH6-abts-1pFH6-abts-4 and the eight SLC26-related plasmids pFH6-sulp-1pFH6-sulp-8. Translational promoter fusions (pTS1-abts-N and pTS1sulp-N) were amplified using 3' genomic primers that annealed immediately before the stop codons to produce in-frame 3' fusions with GFP. Because the abts-4 gene exceeds 17 kb in length, the abts-4 translational fusion was created with the cDNA rather than with the gene. In several instances, rather than clone the promoter/ORF, a PCR product containing GFP from pFH6.II was annealed with a promoter/ORF genomic PCR product with complementary overhangs, and the resulting amplified linear DNA was then injected as described below.
GE24 pha-1(e2123ts) nematodes were cultured at 15°C on nematode growth medium plates containing 5 µg/ml cholesterol seeded with OP50 bacteria from an overnight culture using standard techniques. GFP fusion constructs were mixed with pCL1 at 75 µg/ml each in high-K+ injection buffer to rescue the pha-1 deficit (15) and then were coinjected into the gonad of young adults as described previously (34). After 4 days at 22°C, surviving first generation (F1) progeny from at least 10 injections were picked to separate plates to evaluate germline transmission. F2 generation nematodes anesthetized on 2% agarose pads were irradiated using a 150-W Hg lamp with a TILL Photonics Polychrome IV monochomator and a Nikon Eclipse E2000 microscope with Apo series objectives and a GFP long-pass emission filter set, or using a Leica DRME confocal microscope under 488-nm laser illumination and a fluorescein isothiocyanate filter set as appropriate. Images were processed using Adobe PhotoShop software (Adobe Systems, San Jose, CA).
Anion influx. The ORFs for abts-1, abts-2, sulp-2, and sulp-4 were subcloned into the oocyte expression vector pBF (63). Capped cRNA was transcribed from linearized cDNA templates with SP6 RNA polymerase (Ambion, Austin, TX) and resuspended in diethylpyrocarbonate-treated water. RNA integrity was confirmed by performing agarose gel electrophoresis in formaldehyde. Mature female Xenopus (NASCO, Madison, WI) were maintained and subjected to partial ovariectomy under tricaine hypothermia anesthesia as previously described (20) and in conformity with methods approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. Stages 5 and 6 oocytes were manually defolliculated after incubation of ovarian fragments with 2 mg·ml1 collagenase A or collagenase B (Boehringer Mannheim, Indianapolis, IN) for 60 min in ND-96, pH 7.4, containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (6). Oocytes were injected on the same day with 10 ng of cRNA in 50 nl of water or with the same volume of water. Injected oocytes were then maintained for 26 days at 19°C.
Unidirectional 36Cl influx studies were performed for 60-min periods as previously described in ND-96 at pH 7.4. The [35S]sulfate influx experiments were performed in 150 µl of influx medium containing 5 µCi carrier-free [35S]sulfate (47 nM) in 0.1 mM unlabeled sulfate. The [14C]oxalate influx experiments were performed in Ca2+- and Mg2+-free influx medium containing 0.5 mM oxalate (2.67 µCi·ml1).
Cl/HCO3 exchange measurements.
Oocytes previously injected with abts-1 cRNA or with water were incubated for 30 min at room temperature with 5 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). BCECF-labeled oocytes were mounted in a 0.8-ml superfusion chamber on a microscope stage. Cl/HCO3 exchange was measured using BCECF fluorescence ratio imaging of oocyte pHi changes during removal and restoration of superfusate Cl concentration (72 mM) in the presence of 5% CO2-24 mM HCO3, with gluconate used as the substituting anion (11). Data acquisition and analysis were performed using MetaFluor software (Universal Imaging, Downingtown, PA). Initial rates of dpHi/dt were measured using the least-squares linear fit of initial slopes. Buffer capacity (T) was calculated as the sum of intrinsic buffer capacity (
i), which is 1820 mM/pH unit (59), plus CO2 buffer capacity (
CO2). Data are expressed as proton flux JH+ = dpHi/dt x
T.
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RESULTS |
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The predicted SULP protein sequences are 2535% identical to their mammalian counterparts, with the greatest similarity evident in the hydrophobic predicted transmembrane domains. SULP-3, -4, -5, -7, and -8 polypeptides form a separate subclass within the SULP superfamily, but the low degree of homology with the mammalian SLC26 polypeptides renders ortholog assignment difficult.
All SULP proteins contain STAS domains (3), with the sole exception of the short sulp-7 polypeptide variants. STAS domains of SLC26 proteins may contribute to plasma membrane localization (55), and previous work has shown that loss of this domain abolishes SLC26 transport activity. Loss or inactivating mutation of the STAS domain of SLC26A3 also abolishes stimulation of CFTR in overexpression systems (28). However, unlike the COOH-terminal class I PDZ-binding motifs of the mammalian SLC26 proteins, only SULP-1 among the eight SULP proteins has a predicted (class III) PDZ-binding motif (aa 726729). Additional functionally significant amino acids conserved among mammalian and plant SLC26 proteins (24, 49) are present in the nematode proteins.
Several sulp splice variants were found. Two sulp-1 exon 9 acceptor sites are separated by 12 nt, resulting in optional inclusion of four additional amino acids within a variable loop of the STAS domain (Fig. 1C). A sulp-3 cDNA lacking exon 8 is missing amino acids that are highly conserved within a putative transmembrane domain, but an SLC26A6 splice variant (SLC26A6-d) that contains a similar deletion was recently found to be functional (33), and hAE1 has been shown to retain activity in the absence of transmembrane domains 5 and 6 (16). Sulp-7 gave rise to multiple 5' and 3' RACE products, while the two sulp-8 variants differed at the 5' UTR but encoded identical polypeptides. All sulp transcripts except sulp-3 were found to be SL1 transspliced.
The abts and sulp genes are expressed in both unique and overlapping patterns. Genomic DNA fragments upstream of the mapped start sites for each member of the abts and sulp gene families were cloned into a promoterless GFP expression vector and then injected into nematodes to create transgenic strains that fluoresce in the cells in which each of the transporters is expressed. These expression patterns likely do not reveal the complete cellular range of expression. Strong GFP expression in one cell type may obscure weaker expression in another nearby cell. GFP transgene loss during cell division can result in mosaicism and an incomplete repertoire of fluorescent cells in any given animal. In addition, the expression of transgenes cannot be assumed to replicate accurately native gene expression patterns. However, with these caveats in mind, the gene expression patterns described herein can be used to direct efforts at elucidating physiologically relevant gene function. Table 1 summarizes the cellular patterns of gene expression described below.
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To establish subcellular localization of the putative anion transporter proteins, we expressed translational GFP fusions for abts-2 (single cell), abts-4 (intestine), sulp-2 (intestine), sulp-4 (excretory cell), sulp-5 (excretory cell), and sulp-8 (intestinal and excretory cell). These fusions encompassed a genomic region consisting of the promoter and the entire ORF of the protein, with GFP attached in place of the natural stop codon. In the case of abts-4, the ORF was inserted as a cDNA fragment rather than as a genomic fragment, owing to vector size limitations. Table 2 summarizes the observed localizations of these fusion proteins.
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Anion transport by ABTS and SULP proteins. The polypeptides encoded by the abts and sulp gene families are predicted to function as anion transporters. cRNA transcripts for ABTS-1, ABTS-2, SULP-2, and SULP-4 were injected into Xenopus oocytes. Anion influx assays were performed using Cl, sulfate, and oxalate as substrates. As shown in Fig. 6, SULP-4 exhibited robust transport of sulfate and more modest transport of Cl and oxalate, whereas ABTS-2 did not mediate detectable uptake of Cl, sulfate, or oxalate. The intestinal and neuronal SULP-2 exhibited modest uptake of sulfate. In contrast, the neuronal and hypodermal cell protein ABTS-1 exhibited robust Cl uptake without uptake of sulfate or oxalate (Fig. 6). ABTS-1 was therefore also examined for Cl/HCO3 exchange activity. As shown in Fig. 7, ABTS-1 conferred on Xenopus oocytes a 2.4-fold increase in Cl/HCO3 exchange activity.
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DISCUSSION |
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To establish a genetically tractable model system in which to study AEs in the nematode C. elegans, we generated C. elegans cDNA encoding four abts polypeptides homologous to SLC4 AEs, and eight SULP polypeptides homologous to SLC26 AEs. We defined the cell type-specific expression patterns for these novel C. elegans polypeptides and documented functional anion transport activity for at least one member of each gene family. In combination with our previous studies of C. elegans NHEs, these results provide the foundation for the study of systemic acid-base physiology using the nematode model. Our results suggest that the molecular diversity of mammalian AEs is also present in worms. The four C. elegans abts genes correspond to nine mammalian SLC4 genes. The eight C. elegans sulp genes correspond to 11 SLC26 genes in mammals, one of which appears to be a pseudogene.
The C. elegans abts polypeptide sequences are not clearly orthologous to individual SLC4 sequences, although the highest pairwise percentage amino acid identities were 50% between ABTS-1 and SLC4A8 and 38% between ABTS-3 and the mammalian borate transporter BTR1. BTR1 was recently described as the 11th member of the SLC4 superfamily and was termed NaBC1 to reflect electrogenic Na+-coupled borate transport (43). BTR1 resembled ABTS-2, -3, and -4 more closely than it did any of the other mammalian slc4 isoforms. In addition, while there was a high degree of sequence relatedness between the Na+-HCO3 cotransport arm of SLC4 and ABTS-1, the robust transport of Cl by ABTS-1 expressed in Xenopus oocytes may be more consistent with Na+-dependent Cl/HCO3 exchange, such as that mediated by SLC4A8. In addition, the nematode proteins may display previously unrecognized or specialized functions.
Similarly, individual SULP polypeptides were not clear orthologs of single SLC26 polypeptides or subfamilies. The highest pairwise percentage amino acid identity was 33% between SULP-5 and SLC26A2, although the percentage amino acid similarity was 50% between any given nematode polypeptide and human polypeptide.
Expression of the majority of these transporters was detected in polarized cell types, including the pharynx, the excretory cells, and the cells of the intestine (Table 1). Each of these cell types serves a specialized function in worms, and these functions may suggest physiological roles for these transporters. For example, because no hypodermal layer separates the pharyngeal muscle anchorage from the cuticle and the pharyngeal muscles display myoepithelial properties and participate in the secretion of cuticle, the sulp-3 gene product might facilitate the accumulation of substrates for cuticle formation or in their export from the cell. Among these might be sulfate for sulfate proteoglycans and for the tyrosine sulfation apparently important for cuticular collagen cross linking (25) and (more speculatively) borate for cuticular glycan cross linking.
Laser ablation studies have shown that the excretory cell helps to maintain the osmolarity and volume of the pseudocoelom and that its loss leads to bloating and death within 24 h (40, 41). The expression of at least three sulp isoforms in the excretory cell supports the hypothesis that these SULP proteins enhance transepithelial electrolyte and fluid secretion from the pseudocoelom to the duct lumen.
Furthermore, intestinal expression of multiple sulp genes along with abts-4 highlights the likely importance for this organ of anion transport and perhaps cellular and luminal pH regulation. Because electroneutral NaCl absorption in the mammalian intestine is mediated by parallel Na+/H+ and Cl/HCO3 exchange, it comes as no surprise that targeted genetic deletion of that apical enterocyte NHE3 leads to renal and intestinal absorptive deficits (53), while mutations in human SLC26A3 cause congenital Cl diarrhea (18). Two of the three C. elegans nhx NHEs expressed exclusively in the intestine reside on the enterocyte apical membrane (39). We therefore hypothesized that the enterocyte apical membrane would harbor at least one of the newly identified anion transporter proteins. However, each of the three gene products identified in intestinal cells was localized to the basolateral membrane, and preliminary data suggest that the long isoform of sulp-7 also encodes a protein targeted to the basolateral membrane (data not shown). Indeed, most of the GFP fusion polypeptides were localized basolaterally, which raises the possibility that they might be missorted, especially if normal sorting required integrity of the COOH-terminal PDZ recognition motif. Confirmation of these initial results will require the use of antibodies directed toward the native sequences. It remains possible that the sulp-6 gene product, which could not be examined as a translational GFP fusion because of an incomplete cDNA 3' end, may reside on the enterocyte apical membrane.
We also have shown that SULP-2 resides in the sensory cilia of ADE, PDE, and cephalic neurons. These dopaminergic neurons (62) cooperatively mediate the basal slowing response produced in well-fed worms by the reintroduction of food (52). Dopamine signaling modulates behavioral plasticity in C. elegans (50), and degeneration of these dopaminergic neurons has been studied in a worm model of Parkinson's disease. The restriction of neuronal SULP-2 expression to these three sets of functionally redundant cells suggests a possible role for SULP-2 related to dopaminergic function, and the link between neuronal and intestinal function may be significant. Because the roles of SLC4 and SLC26 AEs in mammalian neuronal function remain little understood (9), the study of C. elegans homologs may provide important insights.
Finally, we have shown that ABTS-2 and SULP-4 each mediate anion transport in Xenopus oocytes. Recombinant expression of ABTS-1 also results in detectable Cl/HCO3 exchange using a microelectrode approach to monitoring membrane voltage and pHi (Romero M, unpublished observations). More extensive study of anion transport selectivity and optimization of functional expression systems will provide further insight to complement ongoing functional genetic studies.
The annotated genome and genetic tractability of the nematode model makes it particularly well suited to the study of integrative physiology (61). The transparency of the nematode allows for the use of transgenic fluorescent fusion proteins for real-time in vivo measurement of protein trafficking, pHi (35), and intracellular cytoplasmic and/or organellar Ca2+ concentration (36). Advances in electrophysiological techniques have provided insights into electrogenic transport processes in the intact worm (14, 46), while the advent of embryonic cell culture has allowed comparative in vivo and in vitro study of identified single cells of defined lineage and developmental status (10). Together with the fast-growing repertoire of defined mutant strains, these experimental advantages facilitate direct physiological and biophysical study of individual ion transporters in the intact living organism. The data presented herein establish a model system for genetic studies of anion transport and acid-base physiology in the intact animal.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The GenBank accession numbers are AY887903AY887917 (abts) and AY954522AY954525 (sulp).
2 During the preparation of this article, a new expressed sequence tag (EST) for sulp-7 that encodes a predicted polypeptide of 582 amino acids, an expected length for a functional sulp family member, appeared in the database.
3 SL1-transsplice leaders were identified for all of the abts gene products, but several EST clones from abts-3 that suggest the existence of two additional 5' exons occurring >10 kb upstream, followed by a large intron and replacing exon 1 (to which the RACE primers were designed), have recently been described in WormBase. This would result in a protein predicted to be 162 amino acids shorter at the 5' end (data not shown). This database information suggests that two alternate promoters may control transcription of the abts-3 gene.
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