INVITED REVIEW
Alternative splicing and diversity of renal transporters

Gerardo Gamba

Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City CP 14000, Mexico


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

The growing molecular identification of renal transporter genes is revealing that alternative splicing is common among transporters. In this paper, I review the physiological consequences of alternative splicing in some genes encoding renal transporters in which spliced isoforms have recently been identified. In some cases, the spliced isoforms resulted in nonfunctional proteins, which, however, possess a dominant negative effect on the cotransporter function, suggesting that the presence of such isoforms can be important in the functional regulation of the transporter. In most transporter genes, however, the spliced isoforms have been shown to be functional, resulting in a variety of physiological consequences, including, for example, changes in the polarization of isoforms to the apical or basolateral membrane, changes in pharmacological or kinetic properties, and changes in tissue distribution or intrarenal localization. In some cases, although the spliced isoform is functional, the consequence of splicing is still unknown. Different regulation among isoforms is an interesting possibility. Thus the diversity of several renal transporters is enhanced by alternative splicing mechanisms.

isoforms; proteome; membrane proteins; tubular reabsorption


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

THE RECENT REPORT OF THE FIRST draft sequence of the human genome (43) predicted the total number of genes in humans to be ~31,000, a number that is consistent with the predicted 35,000 genes in another recent analysis based on a comparison between the human chromosome 22 sequence and the pool of human expressed sequence tag (EST) contigs (22). Thus humans have only twice the number of genes of the worm and fly and six or seven times more than bacteria or yeast, suggesting that increasing complexity in vertebrates is not related to an increased number of genes but to other regulatory mechanisms, including alternative splicing.

Initial reports suggested that ~35% of human genes undergo alternative splicing (32, 50). However, with increased numbers of ESTs in the databases, recent    analyses have shown that alternative splicing occurs in at least 55-60% of human genes (37, 43). The gene-poor chromosome 22 and the gene-rich chromosome 19 exhibit a total of 2.6 and 3.2 distinct transcripts/gene, respectively, with at least 70% of the alternatively spliced forms occurring within the coding regions (43). In addition, the proportion of genes exhibiting alternatively spliced products could be even higher because it has been observed that the frequency of splicing decreases for any gene with >300 EST hits, suggesting that genes with low expression rates, and therefore with the fewer EST hits, are those with higher splicing rates (37). Thus although the human genome contains twice as many genes as worm and fly genomes, it is estimated that, due to alternative splicing, humans probably express five times more protein products.

In eukaryotic cells, alternative splicing mechanisms remarkably increase the coding capacity of the genes. A variety of mRNA transcripts can be produced from a single gene by changing exons in mature mRNA. During mRNA maturation, some exons are always preserved while others are optional. That is, they can or cannot be present. Still other exons are mutually exclusive. That is, one or another exon is included, but not both at the same time. Alternative splicing can also produce changes in the final composition of the protein by introducing a different stop codon and can also change the regulation of mRNA stability by producing transcripts with similar open reading frames but divergent 5'- or 3'-untranslated regions (UTRs). mRNA posttranscriptional processing and modification are complex procedures that include not only alternative splicing but also intron splicing, capping at the 5'-end, and polyadenylation of the 3'-end. Splicing errors can thus be the cause of nonfunctional proteins and, hence, of some hereditary diseases.

Alternative splicing not only allows a single gene to produce a variety of proteins but also can potentially increase the possibilities for regulation of a particular gene product. Some alternative splicing events are constitutive, producing mRNA transcripts that coexist at a constant ratio in the same cells, whereas other splicing events are clearly regulated in response to several physiological or biochemical stimuli. The recognition of a particular splicing site can be enhanced or decreased, resulting in changes in the type and/or proportion of mature mRNA transcripts produced from a single gene (for an excellent review on the regulation of alternative splicing, see Ref. 46). Thus, when several products of a single gene are produced in the same cell, regulation of alternative splicing by external factors can be critical in adjusting the splicing product ratio in relation to physiological stimuli.

Molecular identification in the last decade of several genes encoding renal cotransporters has shown that most of them give rise to alternatively spliced isoforms, resulting in a variety of physiological consequences. Here I present a review of the renal transporter genes in which alternatively spliced isoforms have been recently identified.


    NA+-BICARBONATE COTRANSPORTERS AND EXCHANGERS
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Regulation of intracellular pH is critical to maintaining many cellular processes in optimal conditions. Bicarbonate transport through the cellular membranes is one of the most important mechanisms in the modulation of intracellular pH. In addition, bicarbonate transport in the kidney also plays a critical role in whole body acid-base metabolism. Thus it is not surprising that cells have developed several different bicarbonate transport mechanisms, including cotransporters and exchangers.

At least five genes have been described in the family of Na+-bicarbonate cotransporters and exchangers, known as the NBC family. The first to be identified was the NBC-1 gene from the salamander (72) and mammalian kidney (12). This gene has been localized to human chromosome 4 and gives rise to at least three spliced isoforms. At the NH2-terminal domain, one isoform was cloned from the human kidney (NBC-1A) (12) and another from the human pancreas (NBC-1B) (1) (Table 1). As shown in Fig. 1, both proteins are identical, with 994 amino acid residues featuring 10 putative transmembrane domains, with a hydrophilic loop between domains 5 and 6 containing three glycosylation sites. These isoforms differ at the NH2-terminal domain. NBC-1A is transcribed from an alternative promoter in intron 3 (2), with 41 amino acid residues at the NH2-terminal domain that are not present in the NBC-1B isoform, and is expressed exclusively in the proximal tubular cells in the kidney. In contrast, NBC-1B is transcribed from exon 1 (2), contains 85 distinct amino acid residues at the NH2-terminal end, and is expressed in several tissues. Both are functional without any apparent difference, suggesting that the major consequence of splicing is tissue distribution. In addition, as shown in Fig. 1, there is also a COOH-terminal spliced isoform, rb2NBC-1, that has been described in rat brain (10) (Table 1). This isoform is the result of a 97-bp deletion near the end of the open reading frame, causing a frame shift that changes and extends the end of the open reading frame by 61 amino acid residues at the end of the COOH-terminal domain, which differs from the last 46 residues in the NBC-1B isoform. The rb2NBC-1 isoform is fully functional and is predominantly present in neurons, whereas NBC-1B is predominantly expressed in astrocytes, suggesting that the major consequence of this splicing mechanism is cell distribution within the central nervous system.

                              
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Table 1.   Alternatively spliced isoforms in renal cotransporters



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Fig. 1.   Alternatively spliced isoforms of the Na+-bicarbonate cotransporter (NBC) gene 1. Hydrophobicity analysis predicted the existence of a central hydrophobic domain with 10 putative transmembrane segments (M1-M10), flanked by hydrophilic NH2- and COOH-terminal domains that are presumably located within the cell. There is a glycosylated hydrophilic loop between transmembrane domains 5 and 6. The differences between the isoforms are located at the NH2- or COOH-terminal domains (gray portions). rb, Rat brain.

A cDNA fragment of a second gene encoding a Na+-bicarbonate cotransporter (NBC-2) was identified by Ishibashi et al. (36) from human retina, and, later on, the full-length cDNA was identified by Pushkin et al. (67) from a human muscle library and by Choi et al. (13) from rat aorta. The identity at the amino acid level among these clones is ~90%, and ~50% with the NBC-1 gene. NBC-2 was mapped to human chromosome 3 (68) and codes for a DIDS-insensitive, ethylisopropyl amiloride-inhibitable, electroneutral Na+-bicarbonate cotransporter (13, 67), which, in contrast to NBC-1 in the kidney, has been localized at the basolateral membrane of the outer medulla thick ascending limb cells and intercalated cells of the medullary collecting duct (81). In most tissues, NBC-2 is expressed as a 7.5-kb mRNA transcript, except in testis, in which the transcript is ~4.0 kb. It is not known whether this represents an alternatively spliced isoform.

Amlal et al. (3) identified a 2-kb fragment of the third gene of the family, NBC-3, after screening a human NT-2 cell library with NBC-1 cDNA as a probe. The full-length mRNA of this gene was previously predicted by Nagase et al. (54a) and by Grichtchenko et al. (29) (GenBank accession no. AF069512). At the amino acid level, NBC-3 is 50% identical to NBC-1 and 70% identical to NBC-2. The predicted sequence by Nagase et al. (54a) exhibits 101 extra amino acid residues at the NH2 terminus. NBC-3 encodes for an electrogenic Na+-driven Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, which is robustly expressed in the central nervous system, and with lesser intensity in other tissues (29). Amlal et al. (3) observed that NBC-3 mRNA levels in inner medullary collecting duct cells (IMCD3) are increased several-fold after exposure of these cells to sublethal acid stress (3). These authors also observed that NBC-3 is expressed as three different-size transcripts (9.5, 4.4, and 3 kb) in brain, spinal cord, and adrenal medulla, whereas in all other tissues it is only present as the 4.4-kb transcript. However, as yet no spliced isoforms of NBC-3 have been identified.

A fourth gene of the family was recently identified by Pushkin et al. (69) from a human heart cDNA library and localized in chromosome 2. NBC-4 is a 1,074-membrane protein that exhibits 58, 43, and 67% identity with NBC-1, NBC-2, and NBC-3, respectively. Two alternatively spliced isoforms from this gene have been isolated (70) (Table 1): NBC-4a and NBC-4b with 1,137 and 1,047 amino acid residues, respectively. The difference between these isoforms is at the COOH-terminal domain, due to a 16-bp insertion that shifts the open reading frame, increasing the protein by 90 residues at the end. The consequence of this splicing mechanism on NBC-4 is not known; however, it is likely to play a role in subcellular targeting of the protein because the extra 90 residues in NBC-4a contain two protein-interacting domains (70). In addition, both isoforms are expressed in the heart, whereas only NBC-4a is present in the testis. The function of NBC-4 is still unknown. Finally, Wang et al. (82) have recently identified a fifth gene of the family from an insulin-secreting cell line. The full-length cDNA encodes a 1,088-residue polypeptide that functions as a Na+-driven Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. This new gene is predominantly expressed in brain but also to a lesser extent in several other tissues, including the kidney, and at the protein level exhibits the following degree of identity with NBC members: 50% with NBC-1, 70% with NBC-2, 75% with NBC-3, and 43% with NBC-4.


    PHOSPHATE TRANSPORT
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Phosphate is freely filtrated at the glomerulus and reabsorbed in the proximal tubule, although a small fraction is reabsorbed in the distal tubule. Phosphate reabsorption in the proximal tubule is due to the combination of a Na+-dependent phosphate transport pathway in the apical membrane and by a still not well-defined exit mechanism in the basolateral membrane that probably includes exchange with anions or the existence of a leaky pathway for phosphorus.

Three families of Na+-phosphate transporters have been identified at the molecular level and are named type I, II, and III Na+-phosphate cotransporters. Surprisingly, despite their functional similarities (electrogenic Na+-dependent phosphate transport), there is very little identity among the members of these families. Type I and type II transporters were cloned from mammalian renal mRNA following functional expression strategies in Xenopus laevis oocytes (47, 83), whereas type III was first identified as the receptor for an ape leukemia virus and a mouse amphotropic retrovirus (40, 57). In addition, two homologous genes encode for the type II cotransporter: type IIa (47), located in human chromosome 5, with expression almost restricted to the proximal tubule in mammalian kidney, and type IIb (34), located in human chromosome 4, which is expressed in small intestine and lung (80).

Tatsumi et al. (78) described three isoforms of the rat type IIa cotransporter that appear to be due to alternative splicing (Table 1). Figure 2 shows the proposed topology of the type IIa cotransporter and the three isoforms, NaPi-2alpha , NaPi-2beta , and NaPi-2gamma . NaPi-2alpha cDNA encodes a protein of 337 amino acid residue with high homology to the NH2-terminal domain of the type IIa cotransporter but with 25 amino acids at the end that are completely different. This isoform uses exons 1-8 of the NaPi-2 gene (type IIa), together with part of intron 8 as a coding region and the 3'-UTR. NaPi-2beta cDNA encodes a protein of 327 amino acids, of which the first 174 are identical to the type IIa cotransporter and the remaining 153 residues are quite different, featuring a protein with the first 4 transmembrane domains that lacks the long hydrophilic and glycosylated loop between transmembrane segments 3 and 4. This isoform uses the first five exons of the NaPi-2 cotransporter gene and a new exon that is ~10 kb downstream of exon 13. In contrast, NaPi-2gamma cDNA revealed a protein of 268 amino acid residues completely identical to the COOH-terminal region of the type IIa cotransporter. This isoform uses exons 9-13 of the NaPi-2 gene and intron 8 as the 5'-UTR. Thus the three isoforms represent truncated versions of the type IIa cotransporter. With the use of antibodies directed against the NH2- or COOH-terminal domains of the type IIa cotransporter, bands of expected sizes of these isoforms can be detected in Western blots from brush-border membrane vesicles isolated from rat renal proximal tubular cells, suggesting that these alternatively spliced transcripts are indeed expressed as a protein. None of the three spliced isoforms induced expression of a Na+-dependent phosphate transporter when injected into X. laevis oocytes. However, when coinjected with type IIa transporter cRNA, NaPi-2gamma completely abolished, and NaPi-2alpha partially reduced, the function of the type IIa cotransporter, whereas NaPi-2beta had no inhibitory effect. Therefore, the alpha - and gamma -isoforms exhibit a dominant negative effect that could be important in the functional regulation of the type IIa Na+-phosphate cotransporter.


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Fig. 2.   Alternative splicing in the type IIa cotransporter gene. Left: complete cotransporter, featuring 8 transmembrane domains (M1-M8) with a long hydrophilic loop between transmembrane segments 3 and 4 that exhibits 2 putative glycosylation sites. Right: 3 spliced isoforms that have been identified.


    SULFATE TRANSPORT
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Sulfate is also freely filtrated at the glomerulus and reabsorbed in the proximal tubule by an apical Na+-dependent sulfate transporter (NaSi-1) (48), coupled with an exit pathway in which sulfate is exchanged with anions by the sulfate/oxalate-bicarbonate anion exchanger (Sat-1) (39). NaSi-1 in rats and mice is expressed at the brush-border membrane of the proximal tubule, small intestine, and colon (7, 48), whereas in humans it is restricted to the kidney (45). NaSi-1 expression in the kidney is modulated by factors that regulate sulfate reabsorption, such as vitamin D, sulfate, and potassium intake, thyroid and growth hormone, anti-inflammatory drugs, and heavy metals. The Na+-sulfate cotransporter gene expresses two different-size transcripts in rats and humans and three in mice. Two transcripts of 2.2 and 2.5 kb in rats or mice, and 3.8 and 2.8 kb in humans, are detected by Northern blot analysis of renal total RNA, and it has been shown that both transcripts are derived from an alternative polyadenylation signal, encoding for exactly the same protein (7) (Table 1). The third transcript in mouse is of ~2.1 kb and was obtained only by RT-PCR. This transcript is the result of alternative splicing of exon 2 (7). When injected into X. laevis oocytes, the 2.2- and 2.5-kb cDNAs from mouse induced the expression of a Na+-dependent sulfate transporter, whereas the 2.1-kb cDNA exhibited no functional expression (Table 1). Because exon 2 encodes the second putative transmembrane domain, it is highly likely that its absence precludes the protein from being functional. The physiological role of this spliced transcript is not known. In addition, a second gene encoding a DIDS-resistant, Na+-dependent sulfate cotransporter (SUT-1) has been recently cloned from human endothelial vein cells. SUT-1 protein is 48% identical to NaSi-1 and is expressed mainly in placenta (28).

The sulfate/oxalate-bicarbonate anion exchanger Sat-1 was cloned from rat hepatocytes (11) and is expressed in liver, kidney, muscle, and brain and in proximal tubule is polarized to the basolateral membrane (39). No alternatively spliced isoforms have been described for Sat-1.


    NA+-BILE ACID TRANSPORTER
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Two genes encoding the Na+-dependent bile acid transporter have been identified. They are the liver basolateral sodium-bile transporter (BSBT) (31) and the ileum apical sodium-bile transporter (ASBT) (85). This ASBT gene is also heavily expressed in the proximal tubule (14), in which it plays a critical role for bile acid reabsorption. Two alternatively spliced isoforms from the ASBT gene have been isolated from the rat (44, 76) (Table 1). As shown in Fig. 3, the complete isoform encoding the protein (348 amino acid residues) (76) features an extracellular short NH2-terminal domain containing 2 potential glycosylation sites, followed by 7 putative transmembrane segments, and a shorter isoform of 154 amino acid residues (t-ASBT), which resulted from a 119-bp deletion due to the skipping of exon 2; this produces a protein that contains only the first 3 transmembrane domains, in which the last 29 residues are not present in the longer isoform (44). The physiological relevance of splicing is the polarization and the functional properties of these isoforms. The complete isoform is expressed in the apical membrane of bile acid-transporting epithelia and functions as a Na+-bile acid influx pathway (14), whereas the shorter isoform is expressed on the basolateral membrane and exhibits activity only as a bile acid efflux mechanism (44). It is still unknown whether this shorter isoform is a uniporter or whether it requires Na+ for the transport process. Thus these isoforms are complementary. One does the uptake of Na+-bile acid at the apical membrane, and the other performs the bile acid efflux in the basolateral membrane.


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Fig. 3.   Alternatively spliced isoforms of the apical Na+-bile acid transporter (ASBT). Top: complete cotransporter that is expressed in the apical membrane (ASBT), featuring 7 transmembrane segments (M1-M7), flanked by an extracellular NH2-terminal domain with 2 putative glycosylation sites and an intracellular COOH-terminal domain. Bottom: truncated isoform expressed in the basolateral membrane (t-ASBT) that contains only the NH2-terminal domain and the first 3 transmembrane segments (M1-M3).


    ORGANIC CATION OR ANION TRANSPORTERS
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

The tubular transport of organic substances in the kidney is a fundamental mechanism to secrete unwanted endogenous metabolites, drugs, and environmental chemicals into urine. Proximal tubule cells express several organic anion or cation transporters in both the apical and the basolateral membrane. At least four genes encoding organic anion transporters and five encoding organic cation transporters have been cloned to date. Excellent reviews on these protein families have recently been published (8, 19, 41). Of the five organic cation transporter genes, OCT1 was the first to be identified at the molecular level (30) and is expressed at the basolateral membrane of proximal tubule cells (19). This gene in rat generates two alternatively spliced isoforms that are expressed in kidney, liver, and intestine (91) (Table 1): OCT1, a protein of 556 residues and OCT1a, a protein of 430 residues due to the lack of exon B. As shown in Fig. 4, the consequence of the deletion is that OCT1a lacks the first two transmembrane domains and three putative glycosylation sites in the first extracellular loop. Nevertheless, both proteins are functional, as assessed by [14C]tetraethylammonium uptake in X. laevis oocytes after injection with cRNA from each clone. Functional analysis revealed that affinity of both isoforms for tetraethylammonium is very similar (91). Tissue distribution is also similar between OCT1 and OCT1a. Thus the physiological consequences of the splicing are still unknown.


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Fig. 4.   Two isoforms of organic cation transporter 1 (OCT1). OCT1 has 12 transmembrane domains (M1-M12) and OCT1a that lack the first 2 transmembrane domains and the hydrophilic loop between M1 and M2.


    NA+/H+ EXCHANGER AND NA+-GLUCOSE COTRANSPORTER
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

The Na+/H+ exchanger and the Na+-glucose cotransporter are very important transport pathways for salt, glucose, and acid-base metabolism. At least six genes encode isoforms of the Na+/H+ exchanger, and three genes encode isoforms of the Na+-glucose cotransporters. To the best of my knowledge, however, no alternatively spliced isoforms have been described for these proteins. Excellent reviews about the molecular biology and physiology of these important membrane transporters have been recently published (15, 86).


    NA+/MYO-INOSITOL TRANSPORT
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Myo-inositol is one of the most important intracellular osmolytes that play a critical role in cell volume regulation. This is particularly true for cells in renal medulla that are exposed to a wide fluctuation of extracellular solute concentrations. Medullary cells maintain normal intracellular ion composition, despite the extremely high NaCl and urea concentration of the environment, due to their ability to accumulate osmolytes, such as sorbitol, myo-inositol, betaine, and others. The accumulation of myo-inositol in renal and other cells (i.e., retinal pigment epithelial cells) is due to the function of the Na+-myo-inositol cotransporter (SMIT).

There is only one gene that encodes for the SMIT. Kwon et al. (42) cloned SMIT cDNA from Madin-Darby canine kidney cells following a functional expression strategy in X. laevis oocytes. The SMIT gene is part of the Na+-glucose cotransporter superfamily. In humans, the SMIT gene is located in chromosome 21, within the Down's syndrome region.

The use of SMIT cDNA as a probe revealed the existence of several transcripts within the range of 1.0-13.5 kb in RNA from kidney (42), lens (92), endothelial cells (84), and the central nervous system (59, 84). Some of these transcripts are upregulated by cellular exposure to hypertonicity (42). Interestingly, however, the entire 718 amino acid residues of the SMIT protein are encoded by a single intronless exon (9). Nevertheless, Porcellati et al. (65, 66) have shown that the human SMIT gene is composed by at least five exons, which give rise to several spliced transcripts, of which most exhibit only differences in the 5'- and 3'-UTRs, without apparent functional significance. There are two isoforms, however, that do exhibit changes in the coding region (Table 1). Figure 5 shows the proposed topology for SMIT1, featuring 14 membrane-spanning domains, with a large hydrophilic intracellular domain connecting transmembrane segments 13 and 14 (65). In contrast, the SMIT2 isoform uses an alternatively spliced donor site that is upstream of the exon 2 stop codon, with the same acceptor site on the 5'-end of exon 3, thus generating a new stop codon in exon 4. As a consequence, SMIT2 loses transmembrane domain 14 and exhibits a different hydrophilic domain after membrane segment 13 that is encoded by exons 3 and 4. The other spliced isoform, SMIT3, uses the same alternate donor site as SMIT2, but with a different acceptor site on exon 4, also generating a different stop codon. Thus SMIT3 is also predicted to lack the last transmembrane domain and contains new sequences of the COOH-terminal domain that are encoded by exons 3, 4, and 5. Functional expression studies in X. laevis oocytes have shown that oocytes injected with SMIT1, SMIT2, or SMIT3 cRNA induce the appearance of Na+-dependent myo-inositol transporter activity, exhibiting interesting differences in the response to protein kinase A (PKA) activation. Although SMIT1 is activated by a IBMX-forskolin combination, SMIT2 and SMIT3 are partially inhibited (66). Therefore, alternative splicing of the SMIT gene in humans gives rise to functional isoforms that are differentially regulated by second messengers.


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Fig. 5.   Spliced isoforms of the sodium myo-inositol cotransporter (SMIT1). SMIT1 is encoded entirely by exon 2 and features 14 transmembrane segments (M1-M14). SMIT2 and SMIT3 are identical to SMIT1 until the very end, where membrane segment 14 is absent, and they show unique COOH-terminal sequences that are encoded by exons 3, 4, and 5.


    ELECTRONEUTRAL CLminus -COUPLED COTRANSPORTERS
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Chloride ions are reabsorbed along the nephron by several different mechanisms, one of which is cotransport with monovalent cations in electroneutral fashion. Based on the nature of the cation that is coupled to Cl-, the sensitivity to diuretic inhibitors, and the stoichiometry of the translocation process, four types of electroneutral Cl--coupled cotransporters (CCC) have been identified. The thiazide-sensitive Na+-Cl- cotransporter (TSC) has been specifically localized at the apical membrane of the distal tubule; the loop diuretic-sensitive Na+-K+-2Cl- and Na+-Cl- cotransporters (BSC) are present in the apical membranes of the thick ascending loop of Henle (TALH) and the IMCD basolateral membrane; and the K+-Cl- cotransporter (KCC) has been shown to be present along the entire nephron. Thus the combined function of the CCCs is responsible for 20-30% of the reabsorbed salt and is involved in calcium, potassium, and acid-base metabolism. In addition, the CCCs also serve as the receptors for some of the most potent and frequently used diuretic drugs (thiazide-type and loop diuretics).

Eight genes encoding members of the CCC family have been identified. The first to be cloned was TSC cDNA from winter flounder urinary bladder (25), followed by the cloning of TSC from mammalian sources (24). Mammalian TSC is a protein comprising 1,002 amino acid residues (1,023 in teleosts). In humans, the TSC gene is localized in chromosome 16, and Gitelman's syndrome (hereditary hypokalemic metabolic alkalosis) is the result of mutations in this gene. No isoforms of TSC have been reported in mammals. However, an alternatively spliced isoform with truncation of 229 amino acid residues at the NH2-terminal domain has been shown to be present in several tissues of the winter flounder and other teleosts (25) (Table 1). The physiological consequence of this splicing is still unknown, but the expression of this shorter transcript in teleosts seems to be regulated by water salinity (49).

Two genes encoding the bumetanide-sensitive cotransporter have been cloned from fish and mammalian sources: BSC-1/NKCC2 cDNA, which is expressed only at the apical membrane of the TALH cells (24), and BSC-2/NKCC1, which is present in several cells and tissues (16, 87) including the kidney, where it is present in the basolateral membrane of collecting duct and in vascular smooth cells at the glomerulus. One alternatively spliced isoform lacking exon 21 has been reported for BSC-2/NKCC1 in brain (71). Exon 21 contains a putative PKA phosphorylation site, suggesting that the splicing could result in a Na+-K+-2Cl- cotransporter with different regulatory properties. However, no functional analysis of this isoform has been reported (Table 1).

BSC-1/NKCC2 is localized in chromosome 15, and some of the kindreds with Bartter's syndrome (another hereditary hypokalemic metabolic alkalosis) are the result of mutations in this gene. BSC-1/NKCC2 gives rise to six alternatively spliced isoforms resulting from the combination of two splicing mechanisms (Table 1). As Fig. 6 shows, one of the splicing mechanisms is due to the presence of three 96-bp (31 amino acid residues) mutually exclusive cassette exons (A, B, and F) that encode the putative second transmembrane domain and the connecting segment between transmembrane segments 2 and 3 (35, 60). The other splicing mechanism arises from the utilization of a polyadenylation site in the intron between coding exons 16 and 17, which results in two different COOH-terminal domains (52): a long one with 457 amino acid residues and a short one with 129. Note in Fig. 6 that this splicing also confers differences in putative PKC and PKA phosphorylation sites at the COOH-terminal domain.


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Fig. 6.   Alternatively spliced isoforms of the apical Na+-K+-2Cl- cotransporter gene. Isoforms with the long and short COOH-terminal domain are shown on left and right, respectively. The gray rectangle and the gray connecting segment between M2 and M3 depict the region encoded by the 3 mutually exclusive cassette exons: A, B, and F.  and , Putative protein kinase (PK)C and PKA phosphorylation sites, respectively.

The three isoforms, A, B, and F, exhibit an axial distribution along the TALH. The F isoform is predominantly expressed at the inner stripe of the outer medulla, the A isoform at the outer stripe of the outer medulla, and B isoform at the cortical TALH (35, 60, 89). The three isoforms with long COOH-terminal domain exhibits function as Na+-K+-2Cl- cotransporters (64), and it has been suggested that the difference among the A, B, and F isoforms could be in transport kinetics. We have recently observed that this is indeed the case because the F isoform exhibits significantly lower affinity for Na+, K+, and Cl- than the A and B isoforms (Plata C and Gamba G, unpublished observations).

The shorter isoforms, A, B, and F, with the truncated COOH-terminal domain, contain 55 amino acid residues at the end that are not present in the longer isoforms (52). Interestingly, the shorter isoform is also expressed at the apical membrane of TALH (52) and exerts a dominant negative effect on the function of the Na+-K+-2Cl- cotransporter, which is abrogated by cAMP (64), suggesting that this isoform could be important in the regulation of the Na+-K+-2Cl- function by hormones, such as vasopressin, which generate cAMP via their respective Gs-coupled receptors. In addition, we have recently shown that the shorter isoform codes for a hypotonically activated, loop diuretic-sensitive Na+-Cl- cotransporter that can be inhibited by cAMP or activated by protein kinase A inhibitors (63). In mouse and rabbit TALH, vasopressin and extracellular osmolarity modulate the NaCl transport mode. In the absence of vasopressin and the presence of low tonicity, NaCl is transported in the TALH through a K+-independent, but nevertheless furosemide-sensitive, Na+-Cl- mechanism, whereas the presence of vasopressin or hypertonicity switches the Na+-Cl- transport mode to the furosemide-sensitive Na+-K+-2Cl- cotransporter (21, 77). Therefore, this shorter isoform could provide the explanation for the switch between Na+-Cl- and Na+-K+-2Cl- cotransporters to the TALH.

Four genes encoding isoforms of the K+-Cl- cotransporter have been identified. KCC1, KCC3, and KCC4 are all expressed in the kidney (26, 53), whereas KCC2 is a neuronal-specific isoform (61). The function of the K+-Cl- cotransporters in the kidney has not been defined with precision, but the major possibilities are K+ reabsorption in the proximal tubule (5), K+ secretion in the distal tubule (20), and salt reabsorption or acid-base metabolism in the TALH and collecting duct (4). KCC3 gives rise to two spliced isoforms generated by transcriptional initiation 5' of two separate first coding exons. The longer isoform, KCC3a (53), utilizes exon 1a, whereas KCC3b uses exon 1b, situated 23 kb 3' within the human KCC3 gene on chromosome 15 (54). The predicted KCC3a and KCC3b proteins of 1,150 and 1,099 amino acids, respectively, differ dramatically in content and distribution of predicted phosphorylation sites for protein kinases. Both isoforms are functional. Thus the physiological consequence of this splicing is still unknown.


    UREA
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

Urea is probably the most abundant waste product that the kidney must eliminate from the body. In so doing, urea is concentrated in the renal medullary interstitium to avoid the loss of water that otherwise would accompany the excretion of nitrogenous waste. This concentration contributes to the hypertonicity of the renal medullary interstitium.

Two different genes encoding urea transporters have been identified: the vasopressin-regulated urea transporter gene (UT-A) (75), which is predominantly expressed in the kidney, and the erythrocyte urea transporter (UT-B) (58). These two proteins exhibit 63% sequence identity, and in humans both genes are located in the same locus on chromosome 18. In addition to erythrocytes, the product of the UT-B gene is also present in the kidney, but the expression is limited to the endothelial cells of the vasa recta in the inner medulla and the inner part of the outer medulla (88).

As Fig. 7 shows, five different alternatively spliced isoforms of the mammalian UT-A gene have been identified and named: UT-A1 (75), UT-A2 (90), UT-A3 (38), UT-A4 (38), and UT-A5 (23) (Table 1). UT-A1, UT-A3, and UT-A4 exhibit the same transcription star site (6). UT-A1 is the complete isoform of the urea transporter, whereas the other four are truncated isoforms. Nevertheless, all five induced the expression of a phloretin-sensitive urea transport pathway when injected into X. laevis oocytes or transiently transfected into HEK-293 cells. Rat UT-A1 is a protein with 929 amino acid residues and a calculated mass of 102 kDa. The transporter consists of two halves, each containing six putative hydrophobic membrane-spanning domains and a putative extracellular hydrophilic loop, with at least one glycosylation site between transmembrane segments 3 and 4. The two halves are connected by a hydrophilic loop with several putative PKA and PKC, and one tyrosine, phosphorylation sites. UT-A1 is predominantly expressed at the renal inner medulla as a 4.0-kb transcript (75), and immunolocalization has revealed that UT-A1 is expressed exclusively at the apical membrane of IMCD cells (55).


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Fig. 7.   Topology and spliced forms of the UT-A urea transporter gene. The longest isoform is UT-A1, depicted in the center. The other isoforms are truncated versions of UT-A1. UT-A2 is basically the COOH-terminal half of UT-A1, whereas UT-A3 is the NH2-terminal half of UT-A1. UT-A4 is made from the NH2-terminal third of UT-A1 spliced into the two-thirds of COOH terminal of UT-A1. UT-A5 is identical to UT-A3, except for the absence of most of the NH2-terminal domain. Symbols are defined as described in Fig. 6.

The rabbit UT-A2 transporter is 397 amino acid residues in length, corresponding to the CCOH-terminal half of UT-A1 (Fig. 7), and is expressed in the descending thin limb of Henle's loop (90). In contrast to the other UT-A spliced isoforms, the function of UT-A2 is not affected by cAMP or forskolin (90). Rat UT-A3 is a 460-amino acid peptide that is basically the NH2-terminal half of UT-A1, whereas rat UT-A4 is a 466-residue protein corresponding to the first quarter of UT-A1, spliced into the last quarter of UT-A1 (38). UT-A3 protein has also been immunolocalized only to the apical membrane of the IMCD (79). The exact localization of UT-A4 expression is not known. Finally, an even shorter isoform of 323 amino acid residues has been identified from mouse testis and named UT-A5 (23). The amino acid sequence is 100% identical to UT-A3 but lacks the first 139 amino acid residues, with a distinct 5'-UTR. UT-A5 is expressed only in testis, where it has been localized in the peritubular myoid cells surrounding the seminiferous tubules. UT-A5 is the smallest of the UT-A isoforms that is functional. In addition to differences in function and localization, the UT-A alternatively spliced isoforms also exhibit differences in response to regulatory stimuli. For example, water restriction or vasopressin administration increases UT-A2 mRNA levels, whereas no effect is observed in UT-A1 mRNA (for review, see Refs. 73 and 74).


    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

A growing number of gene encoding membrane transporters in the kidney give rise to alternatively spliced isoforms. Most of the time, these isoforms are functional, resulting in a variety of physiological effects. For example, two spliced isoforms of the Na+-acid bile transporter (Fig. 3) exhibit different polarization to the apical or basolateral membrane. The shorter isoform lacks the last four putative membrane-spanning domains and nevertheless is functional but exhibits different functional properties. The apical Na+-K+-2Cl- cotransporter gene gives rise to six alternatively spliced isoforms (Fig. 6) that also confer different functional properties. Three isoforms with the long COOH-terminal domain function as a Na+-K+-2Cl- cotransporter but with different affinity for ions and differential expression along the TALH, whereas the three isoforms with the short COOH-terminal domain function as a Na+-Cl- cotransporter that is hypotonically activated and inhibited by cAMP. The urea transporter gene UT-A produces five different spliced forms (Fig. 7) that show different intrarenal distribution and response to second messengers. In some other transporters, spliced isoforms are functional without any apparent changes in their properties, despite the effect of splicing on the secondary structure of the protein. For example, the shorter isoform of the organic cation transporter (Fig. 4) lacks the first two transmembrane domains, together with an extracellular loop that is probably glycosylated. However, the functional properties of this isoform are the same as those of the longer isoform. A similar observation has been shown to occur for the isoforms of the Na+-bicarbonate cotransporter 1 (Fig. 1). In these examples, the physiological consequences of the splicing are still unknown, and thus a number of testable hypotheses can be suggested, including different regulation by second messengers. In addition, there are genes in which nonfunctional spliced isoforms have been shown to possess a dominant negative effect on the cotransporter function, suggesting an interesting mechanism of protein function regulation. The Na+-phosphate and the Na+-K+-2Cl- cotransporters produce truncated isoforms with a dominant negative effect on the function of the cotransporters, and in the Na+-K+-2Cl- cotransporter the dominant negative effect of the truncated isoform can be modulated by cAMP. One interesting example of dominant negative regulation occurs in the human KvLQT1 K+ channel. This gene is responsible for the congenital long QT syndrome and produces two alternatively spliced isoforms. One forms the functional K+ channel, and the other is a COOH-terminal truncated isoform that does not exhibit function as a channel but exerts a strong dominant negative effect on channel function (17). It has been shown in patients with the recessive form of the long QT syndrome (Jervell and Lange-Nielsen syndrome) that mutation in the dominant negative isoform correlates with the phenotype of cardiac arrhythmia (51) as well as that transgenic mice overexpressing the dominant negative isoform develop several interesting cardiac arrhythmias (18). The mechanisms by which the dominant negative isoforms exert their effect, however, are still poorly understood. The formation of heterodimers between isoforms and the competition between intracellular vesicles containing different isoforms have been suggested as possible mechanisms. However, to the best of my knowledge no study has addressed this issue in membrane transporters. A recent study showed in the glucocorticoid receptor that formation of nonfunctional heterodimers seems to be at least part of the mechanism by which the dominant negative isoform reduces the function of the receptor (56).

The identification of alternatively spliced isoforms in renal transporters has opened a new window for research in molecular physiology. Once we understand the purpose of each splicing, we will be able to analyze the regulation of the splicing mechanisms to know how the cells decide which isoform to produce, how much of each isoform is to be produced at a particular moment, and how this is regulated by intracellular or extracellular physiological stimuli.


    ACKNOWLEDGEMENTS

I am grateful to all members, past and present, of the Molecular Physiology Unit for enthusiastic work and stimulating discussions.


    FOOTNOTES

The author is an International Scholar of the Howard Hughes Medical Institute. The work performed in the author's laboratory was possible thanks to support from the Consejo Nacional de Ciencia y Tecnología, Dirección General del Personal Académico of the National University of Mexico, Fundación Miguel Alemán, and Howard Hughes Medical Institute and National Institutes of Health Grants 75197-553601 and DK-36803, respectively.

Address for reprint requests and other correspondence: G. Gamba, Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, México City, Mexico (E-mail: gamba{at}conacyt.mx).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
NA+-BICARBONATE COTRANSPORTERS...
PHOSPHATE TRANSPORT
SULFATE TRANSPORT
NA+-BILE ACID TRANSPORTER
ORGANIC CATION OR ANION...
NA+/H+ EXCHANGER AND NA+-...
NA+/MYO-INOSITOL TRANSPORT
ELECTRONEUTRAL CL--COUPLED...
UREA
CONCLUSIONS
REFERENCES

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