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
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.
 |
INTRODUCTION |
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 |
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.
View larger version (27K):
[in this window]
[in a new window]
|
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
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
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 |
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-2
, NaPi-2
, and NaPi-2
. NaPi-2 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-2 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-2 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-2 completely
abolished, and NaPi-2 partially reduced, the function of the type
IIa cotransporter, whereas NaPi-2 had no inhibitory effect.
Therefore, the - and -isoforms exhibit a dominant negative effect
that could be important in the functional regulation of the type IIa
Na+-phosphate cotransporter.
View larger version (19K):
[in this window]
[in a new window]
|
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 |
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 |
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.
View larger version (22K):
[in this window]
[in a new window]
|
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 |
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.
View larger version (28K):
[in this window]
[in a new window]
|
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 |
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 |
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.
View larger version (39K):
[in this window]
[in a new window]
|
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 CL -COUPLED COTRANSPORTERS |
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.
View larger version (28K):
[in this window]
[in a new window]
|
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 |
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).
View larger version (45K):
[in this window]
[in a new window]
|
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 |
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.
I am grateful to all members, past and present, of the Molecular
Physiology Unit for enthusiastic work and stimulating discussions.
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).
TOP
ABSTRACT
INTRODUCTION
-COUPLED...
