1 Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City; and 2 Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Coyoacán 04510, Mexico City, Mexico
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ABSTRACT |
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The purpose of the present study was to
determine the major functional, pharmacological, and regulatory
properties of the flounder thiazide-sensitive Na-Cl cotransporter
(flTSC) to make a direct comparison with our recent characterization of
the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161-F169, 2000).
When expressed in Xenopus laevis oocytes, flTSC exhibits
lower affinity for Na+ than for Cl, with
apparent Michaelis-Menten constant (Km) values
of 58.2 ± 7.1 and 22.1 ± 4.2 mM, respectively. These
Km values are significantly higher than those
observed in rTSC. The Na+ and Cl
affinities
decreased when the concentration of the counterion was lowered,
suggesting that the binding of one ion increases the affinity of the
transporter for the other. The effect of several thiazides on flTSC
function was biphasic. Low concentrations of thiazides
(10
9 to 10
7 M) resulted in activation of
the cotransporter, whereas higher concentrations (10
6 to
10
4 M) were inhibitory. In rTSC, this biphasic effect was
observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by
the Na+ or the Cl
concentration in the uptake
medium. In addition to thiazides, flTSC and rTSC were inhibited by
Hg2+, with an apparent higher affinity for rTSC. Finally,
flTSC function was decreased by activation of protein kinase C with
phorbol esters and by hypertonicity. In summary, we have found
significant regulatory, kinetic, and pharmacological differences
between flTSC and rTSC orthologues.
metolazone; distal tubule; osmolarity; salt reabsorption; thiazide-sensitive sodium-chloride cotransporter
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INTRODUCTION |
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EARLY WORK IN THE
MARINE teleosts starry flounder (Platichithys
stellatus) (22) and oyster toadfish (Opsanus
tau) (23) suggested the hypothesis that the teleost
bladder changed the final ion composition of urine by active
reabsorption of ions. Then, after the observation that the isolated
urinary bladder from the marine teleost sea raven (Hemitripterus
americanus) exhibited active absorption of both Na+
and Cl ions (14), Renfro et al. (36,
37) demonstrated in the urinary bladder of the winter flounder
(Pseudopleuronectes americanus) the existence of an active
and interdependent pathway for Na+ and Cl
absorption in the apical membrane (36, 37), together with localization of the Na+-K+-ATPase in the
basolateral membrane (38). Thus the model suggested for
salt reabsorption in the bladder included an apical Na-Cl cotransporter. Years later, Stokes et al. (41) reported
clear evidence that the Na-Cl cotransporter in the apical membrane of the winter flounder urinary bladder was inhibited by the thiazide-type diuretics hydrochlorothiazide and metolazone, and Ziyadeh et al. (47) observed that inhibition of this cotransporter in the
urinary bladder with thiazides was followed by an increase in
Ca2+ absorption. Therefore, ion reabsorption mechanisms in
the teleost urinary bladder and mammalian distal convoluted tubule
(DCT) are similar. We thus took advantage of this similarity to clone a cDNA that encodes the flounder thiazide-sensitive Na-Cl cotransporter (flTSC), using a functional expression strategy in Xenopus
laevis oocytes (11), followed by the identification
of cDNA in the mammalian TSC (rTSC) with a homology-based approach
(10, 20, 40).
TSC belongs to the superfamily of electroneutral cation-coupled
Cl cotransporters from which eight genes have been
identified: two that encode the bumetanide-sensitive Na-K-2Cl
cotransporter (10, 45); one that encodes the
thiazide-sensitive Na-Cl cotransporter (10); four genes
that encode the K-Cl cotransporters (13, 29, 32); and one
gene that encodes a membrane protein with a function that is still
unknown (1). In humans, the gene of the thiazide-sensitive
Na-Cl cotransporter is localized in chromosome 16. Several
mutations in TSC have been associated with an autosomal recessive
disease featuring hypokalemic metabolic alkalosis, chronic arterial
hypotension, hypomagnesemia, and hypocalciuria, known as Gitelman's
syndrome (40), and some of these mutations have been shown
to preclude the protein to be functional due to disruption of the
adequate intracellular processing of the transporter (20).
Little is known about the structure-function relationships of the thiazide-sensitive cotransporter. The identity between the human and shark basolateral Na-K-2Cl cotransporters is ~74%, and the ion and bumetanide inhibition kinetics are very different. For instance, the affinity for the three cotransported ions and for bumetanide of the shark's Na-K-2Cl cotransporter is remarkably lower than the affinity of the human cotransporter. The similar structure but different functional properties between species allowed Isenring et al. (16, 17) to construct and guide experiments using chimeric clones between the human and shark proteins that have been useful in finding out the membrane-spanning domains that determine the ions and bumetanide affinities. Similarly, rTSC and flTSC also show a high degree of homology. The identity is 62% at the amino acid level, plus 10% of conservative changes. Moreover, comparison of our recent analysis of the functional and pharmacological properties of rTSC protein as expressed in X. laevis oocytes (28) with the initial characterization of flTSC (11) suggested that important differences in TSC functional properties could occur among species. Thus the major goal of the present study was to determine the major functional properties of flTSC, also expressed in X. laevis oocytes, to make a direct comparison with rTSC functional properties. In addition, in the present study we extended our previous characterization of rTSC. As a result, here we show important functional, pharmacological, and regulatory differences between rTSC and flTSC, which, together with our present knowledge of the primary structure of both proteins, can be used to begin to explore the structure-function relationships of this cotransporter.
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METHODS |
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X. laevis oocyte preparation.
Oocytes surgically harvested from adult female X. laevis
frogs (Nasco, Fort Atkinson, MI) anesthetized by immersion in 0.17% tricaine were incubated for 1 h under vigorous shaking in frog Ringer-ND-96 [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl,
and 5 HEPES/Tris, pH 7.4] in the presence of 2 mg/ml of collagenase. Then, oocytes were washed four times in ND-96, manually defolliculated, and incubated at 18°C overnight in ND-96 with 2.5 mM sodium pyruvate and 5 mg/100 ml of gentamicin. The next day, the mature oocytes [stages V-VI (7)] were injected with 50 nl of a 1 mM
Tris solution containing either flTSC or rTSC cRNA at a concentration
of 0.5 µg/µl. Control oocytes were injected with the same solution
without cRNA. Oocytes were incubated for 3-4 days in ND-96 with
sodium pyruvate and gentamicin. The incubation medium was changed every 24 h. The night before uptake experiments, oocytes were incubated in Cl-free ND-96 [(in mM) 96 Na+
isethionate, 2 K+-gluconate, 1.8 Ca2+-gluconate, 1.0 Mg2+-gluconate, 5 mM HEPES,
and 2.5 sodium pyruvate, plus 5 mg% gentamicin, pH 7.4]
(10).
In vitro flTSC or rTSC
cRNA translation.
flTSC or rTSC cDNAs were linearized at the 3'-end using NotI
(Life Technologies, Bethesda, MD), and cRNA was transcribed in vitro, using a T7 RNA polymerase mMESSAGE kit (Ambion).
Transcription product integrity was confirmed on agarose gels, and
concentration was determined by an absorbance reading at 260 nm (DU
640, Beckman, Fullerton, CA). cRNA was stored frozen in aliquots at
80°C until use.
Assessment of the Na-Cl cotransporter function.
Functional analysis of the Na-Cl cotransporter was assessed by
measuring the uptake of tracer 22Na+ (New
England Nuclear) in groups of at least 15 oocytes, using our previously
published protocol (28). In brief, oocytes were exposed to
a 30-min incubation period in an isotonic K+- and
Cl-free medium [(in mM) 96 Na+-gluconate,
6.0 Ca2+-gluconate, 1.0 Mg2+-gluconate, 5 HEPES/Tris, pH 7.4] with 1 mM ouabain, 100 µM bumetanide, and 100 µM amiloride, followed by a 60-min uptake period in a K+-free isotonic medium. For most experiments, the isotonic
medium contained (in mM) 96 NaCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4, supplemented with 1 mM ouabain,
100 µM bumetanide, 100 µM amiloride, and 2.0 µCi of
22Na+. All uptakes were performed at 30°C. At
the end of the uptake period, oocytes were washed five times in
ice-cold uptake solution without isotopes to remove extracellular fluid
tracer. After the oocytes were dissolved in 10% sodium dodecyl
sulfate, tracer activity was determined for each oocyte by
-scintillation counting.
Statistical analysis. Kinetic parameters were estimated by nonlinear regression fit of the uptake rate data to the Michealis-Menten (Km) equation. Statistical significance was defined as two-tailed P < 0.05, and the results are presented as means ± SE. The significance of the differences between groups was tested by one-way ANOVA with multiple comparison using Bonferroni correction or by Kruskal-Wallis one-way ANOVA on ranks with Dunn's method for multiple comparison procedures, as needed.
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RESULTS |
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Figure 1 depicts a representative
experiment showing that water-injected X. laevis oocytes do
not express a thiazide-sensitive Na-Cl pathway (10, 28),
whereas oocytes injected with flTSC cRNA exhibited an increased
22Na+ uptake that was sensitive to the
thiazide-type diuretic metolazone and was completely abolished in the
absence of extracellular Cl. As shown in the
inset in Fig. 1, the increased 22Na+
uptake in flTSC cRNA-injected oocytes was linear, at least during the
first 15 min of exposure to the tracer Na+.
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Figure 2 shows the
22Na+ uptake in flTSC-injected oocytes exposed
to different extracellular pH values, in a range from 6.0 to 8.0. For
this experiment, oocytes were exposed to the tracer Na+ in
uptake media containing 80 mM NaCl, titrated to pH of 6.0, 6.5, 7.0, 7.5, and 8.0, with HCl or NaOH, respectively. The uptake rate, the
degree of Cldependency, and the sensitivity to the
thiazide-type diuretic metolazone were similar in all groups (not
significant using 1-way ANOVA), suggesting that extracellular pH had no
effect on flTSC function or on metolazone-induced inhibition of the
cotransporter.
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Kinetic properties of flTSC.
To determine the kinetic properties of flTSC, we assessed
22Na+ uptake in flTSC cRNA-injected oocytes as
a function of the concentration of each transported ion. The results of
these experiments are shown in Fig. 3,
A and B. Uptake experiments were performed with Na+ or Cl fixed at 96 mM and changing
concentrations of the counterion from 0 to 80 or 96 mM. The small mean
values of uptake for water-injected oocytes (data not shown) were
subtracted from those corresponding to flTSC cRNA-injected groups.
22Na+ influx in flTSC showed saturation
kinetics, with estimated Km and maximal velocity
(Vmax) values for extracellular Na+
of 58.2 ± 7.1 mM and 33,670 ± 2,239 pmol · oocyte
1 · h
1,
respectively (Fig. 3A), and for extracellular
Cl
of 22.1 ± 4.2 mM and 25,820 ± 2,022 pmol · oocyte
1 · h
1,
respectively (Fig. 3B).
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Effect of thiazides and
Hg2+ on
flTSC.
The inhibition of the Na-Cl cotransporter by thiazide-like diuretics is
one of the distinctive features of this cotransporter pathway in the
teleost urinary bladder and the apical membrane of the mammalian DCT.
For this reason, we analyzed the effects of increasing concentrations
of several thiazide-type diuretics on the functional expression of the
cotransporter on flTSC cRNA-injected oocytes. Figure
4 shows the results of these series of
experiments, and Table 1 shows the
percentage of Na+ uptake by flTSC in the presence of the
different concentrations of each of the thiazide-type diuretics.
Interestingly, the effect of the thiazide-type diuretics on flTSC
function was biphasic. At low concentrations (106 to
10
9 M), thiazides induced an increase in Na+
uptake that was statistically significant compared with the uptake in
the absence of thiazide. At higher concentrations, 10
6 to
10
4 M, all thiazides reduced the Na+ uptake
through the Na-Cl cotransporter. The inhibitory profile for flTSC
inhibition was metolazone > polythiazide > bendroflumethiazide > trichloromethiazide > chlorthalidone.
This profile was similar to the profile observed for rTSC
(28) but with lower sensitivity. In fact, at a
10
4 M concentration, trichloromethiazide and
chlorthalidone reduced the function of flTSC by only 68 and 46%,
respectively, whereas in rTSC the same concentration of all thiazides
inhibited the function of the cotransporter by >95%
(28). Thus flTSC exhibits lower affinity for thiazides
than does rTSC. The biphasic effect was not observed for most thiazides
in rTSC (28), with the exception of chlorthalidone, which
is the thiazide with the lowest inhibitory potency. The effect of
increased concentrations of chlorthalidone on rTSC function is shown in
Fig. 5. Between 10
9 and
10
7 M concentration, the function of rTSC was
significantly increased. The increased Na+ uptake with the
low concentration of thiazides was Cl
dependent and
sensitive to 10
4 M polythiazide (data not shown),
indicating that it was secondary to stimulation of the Na-Cl
cotransporter function. At 10
6 M chlorthalidone, rTSC
function was similar to that in the absence of thiazide, and higher
concentrations inhibited the function of the rat cotransporter.
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Regulation of flTSC.
The function of all members of the electroneutral cotransporter family
is regulated by cell volume. For example, BSC2 is activated by
hypertonicity and inhibited by hypotonicity (6). BSC1
express a long COOH-terminal isoform that is activated by hypertonicity and partially inhibited by hypotonicity (10), and a short
COOH-terminal isoform that is activated by hypotonicity
(34). The four KCC isoforms are activated by cell swelling
(27, 29). In addition, we have recently shown that rTSC is
also inhibited by cell swelling (28). Thus we analyzed the
effect of exposing oocytes to hypotonic or hypertonic media on the
function of flTSC. For these experiments, oocytes were exposed to an
uptake medium containing 40 mM NaCl or Na-gluconate at three different
osmolarities [hypotonic (~110 mosmol/kgH2O), isotonic
(~210 mosmol/kgH2O), or hypertonic (~310 mosmol/kgH2O, by adding sucrose to the 40 mM NaCl uptake
medium)]. Therefore, uptake rates were determined in different osmolar
conditions, without a change in the extracellular NaCl concentration or
ionic strength. As shown in Fig. 8, when
oocytes were incubated in hypotonic and in isotonic media, the function
of flTSC was similar. However, when oocytes were exposed to
hypertonicity, the Na+ uptake rate by flTSC was
significantly reduced.
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DISCUSSION |
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In the present study, we determined the major functional,
pharmacological, and regulatory properties of the thiazide-sensitive Na-Cl cotransporter that is expressed in the apical membrane of the
winter flounder urinary bladder. As shown before (11),
microinjections of X. laevis oocytes with flTSC cRNA induce
the appearance of a Na+ uptake mechanism that is
Cl dependent and sensitive to thiazide-type diuretics.
The increased Na+ uptake is not sensitive to inhibitors of
other electroneutral cotransporter systems such as furosemide or
acetazolamide (11). In the present study, we observed a
number of interesting similarities and differences between rTSC
(28) and flTSC.
Tran et al. (42) observed that external pH modulated the
[3H]metolazone binding to rat renal cortical membranes.
Maximum [3H]metolazone binding was observed at
extracellular pH of 5.5, followed by a slight decrease, as the pH was
increased up to 8.0, suggesting that external pH, within the range of
changes that can be observed along the DCT, could affect binding of the
thiazide-type diuretics to the cotransporter. However, our results in
flTSC in the present study, comparable to those observed in rTSC
(28), revealed that extracellular pH within the range of
6.0 to 8.0 has no effect on either cotransporter function,
Cl dependency, or thiazide sensitivity. Similar to our
observations in flTSC and rTSC, the function of another member of the
electroneutral cation chloride cotransporter, the K-Cl isoform KCC1, is
not affected by external pH (24).
Analysis of flTSC ion transport kinetics revealed
Km values for extracellular Na+ and
Clat physiological concentrations of the counterion of
~58 and ~22 mM, respectively. Similar values were obtained when
uptake rates were measured using 90- or 15-min incubations. These
results are significantly higher than those obtained by us in rTSC,
also using the X. laevis expression system, which were ~6
mM for both Na+ and Cl
(28).
Therefore, the affinity for the cotransported ions is lower in the
flounder than in the rat cotransporter. In addition, flTSC exhibits
higher affinity for Cl
than for Na+, whereas
in the rat cotransporter the affinity for both ions is similar
(28). The order of binding, however, is comparable between
rTSC and flTSC. We observed in flTSC that the affinity for
Na+ or Cl
, as well as the apparent
Vmax, was reduced when the concentration of the
counterion used in the uptake media was lowered. Although more
concentrations need to be used in the kinetic experiments for better
estimates of the kinetic parameters, the main kinetic features observed
in flTSC with the use of two concentrations of the counterion, below
and above Km, are similar to those previously observed by us for rTSC, using more concentrations (28).
In a rapid-equilibrium, random mechanism, the apparent
Vmax values are dependent on the concentration
of the counterion kept constant during the experiment. That is, the
apparent Vmax should decrease as the fixed
counterion does. Therefore, we believe that our data are consistent
with a rapid-equilibrium, random addition of the ions to the
cotransporter. The other mechanism that could explain our data is an
ordered one in the steady state. However, given that the cotransport of
ions across a membrane most likely does not take place under
steady-state conditions, because the rate of movement of ions is much
slower than the rate of the association and dissociation steps, we
favor a rapid-equilibrium, random mechanism over an ordered
steady-state mechanism. Interestingly, however, although
Na+ and Cl
interaction in flTSC is similar to
that in rTSC (28), we observed no interaction between ions
and metolazone in flTSC. The inhibitory kinetics of metolazone in flTSC
were similar when the concentration of both ions was 96 mM and when
either Na+ or Cl
was used at a very low
concentration (Fig. 6). In contrast, we observed in rTSC that lowering
the extracellular Na+ or Cl
concentration
increased the apparent affinity of the cotransporter for metolazone,
suggesting that in rTSC both ions compete or interact with the diuretic
(28). Therefore, our present data suggest that this
interaction is not present in flTSC.
flTSC is specifically inhibited by thiazide-type diuretics, but the
affinity of the cotransporter for these compounds is lower than the
affinity shown by rTSC (28). In fact, even at
104 M concentration of some thiazides like chlorthalidone
or trichloromethiazide, the inhibition of flTSC was not complete,
whereas in rTSC this concentration of any thiazide inhibited the
cotransporter by >95% (28). In addition, a biphasic
response to thiazides was observed in flTSC. Lower concentrations were
often associated with an increase in the Na+ uptake,
whereas higher concentration reduced Na+ uptake. This
behavior was observed in rTSC only for chlorthalidone, the thiazide
with the lower potency. We believe that the increase in Na+
uptake when oocytes were exposed to low concentrations of thiazides was
due to stimulation of the cotransporter function because the increased
uptake was Cl
dependent and completely sensitive to a
10
4 M concentration of polythiazide.
It has been suggested by several authors that inhibition of the
cotransporter by thiazide is due to the direct interaction of the drug
with the cotransporter, presumably due to competition of the diuretic
with Cl for the same site on the transporter
(42), or with both Na+ and Cl
(28). The increase in cotransporter function, however, is
unlikely to result from such a direct interaction. Instead, it is
possible that thiazides activate a second messenger pathway within the oocytes that probably results in activation of the cotransporter. This
effect is lost with the higher concentrations of the thiazide because,
regardless of the activation of any intracellular pathway, at high
concentrations the thiazide blocks the function of the cotransporter
itself. It has been suggested for years that thiazides reduce blood
pressure not only because of their diuretic and saliuretic action but
also by reducing the peripheral resistances due to a direct
vasodilatory action (4, 5). Some thiazide derivatives, like indapamine or diazoxide, are vasodilators (39) and
not diuretics, and several authors have shown direct effects of
hydrochlorothiazide on blood vessels (2). Thiazides
activate ion channels in vascular smooth muscle cells
(33), and a recent study shows that the mechanism by which
thiazides induce vasodilatation requires endothelium and is inhibited
by the nitric oxide synthesis inhibitor
N
-nitro-L-arginine, suggesting
that the nitric oxide pathway could be implicated (3).
Moreover, in the central nervous system it has been shown that
cyclothiazide modulates the desensitization of the AMPA and kainate
receptors by a mechanism that includes the nitric oxide/cGMP pathway
(9). This effect of cyclothiazide on glutamate and kainate
receptors has been shown to be present when X. laevis
oocytes were used as the expression system (31), suggesting that the required intracellular pathway is present in
oocytes. Thus it is possible that thiazides activate a second messenger
pathway within oocytes that, in turn, results in TSC activation.
However, we observed no effect of cAMP, cGMP, or IBMX on flTSC
function, whereas PKC activation with a phorbol ester resulted in
significant reduction of the cotransporter function. This inhibitory
effect of protein kinase C activation was also observed in rTSC
(15). Further studies will be necessary to elucidate the
intracellular pathway involved.
Hg2+ was used in the first half of the twentieth century as
the first potent diuretic agent available in clinical medicine
(8). The prescription of Hg2+ was then
discontinued due to the toxicity and tendency toward tachyphylaxis,
together with the development of better diuretics such as thiazides and
loop diuretics. The site of action in the nephron was localized at the
thick ascending limb and distal nephron, regions in which
Hg2+ inhibited net Cl reabsorption
(46). However, the precise mechanism of action was never
determined. We show here that Hg2+ reduces the function of
rTSC and flTSC (Fig. 7). The inhibition of flTSC by Hg2+
was observed by Wilkinson et al. (44) using isolated
sheets of the flounder urinary bladder and also by Jacoby et al.
(18) on the basolateral isoform of the Na-K-2Cl
cotransporter. In addition, Hg2+ also inhibits the function
of the apical renal-specific isoform of the Na-K-2Cl cotransporter
(Plata C and Gamba G, unpublished observations). Thus it is possible
that the diuretic effect of Hg2+ was due to direct
inhibition of the Na-K-2Cl and the Na-Cl cotransporters located at the
apical membrane of the thick ascending loop of Henle and the distal
tubule, respectively.
The function of all members of the electroneutral cotransporter family is regulated by cell volume. For example, the Na-K-2Cl cotransporter is activated by cell shrinkage (45), whereas the K-Cl cotransporters are activated by cell swelling (27). In any case, the increase or decrease in the activity of the cotransporter is related to phosphorylation and dephosphorylation processes. TSC was less known as a cell volume-regulated cotransporter. However, when expressed in X. laevis oocytes, rTSC function, compared with isotonicity, is partially reduced in the presence of a hypotonic medium, whereas hypertonicity has no further effect on the cotransporter function (28). Interestingly, flTSC exhibits the opposite behavior. When expressed in X. laevis oocytes, the function of flTSC was similar in hypotonicity and isotonicity, whereas exposure of oocytes to hypertonicity resulted in a significant reduction of cotransporter function.
The urinary bladder in teleosts is produced by the fusion and expansion
of the archinephric ducts and thus constitutes an epithelium that is
embryologically similar to the mammalian DCT (21). The
urinary bladder and the DCT provide a mechanism to reduce amounts of
salt and water and concentrate divalent cations in the urine, in an
environment in which tubular fluid is often more diluted than plasma
(19). Thus we believe that differences in TSC
properties in the flounder and rat are more related to their primary
structure than to the intraluminal milieu. Figure 10 depicts the proposed topology for
TSC. There is a central hydrophobic segment containing 12 putative
membrane-spanning domains that are flanked by a short NH2-
and a long COOH-terminal domain. The hydrophilic segment that connects
membrane-spanning domains 7 and 8 is glycosylated
and thus located outside the cell (30). Although this
topology has not been experimentally confirmed for TSC, it has been
shown to be the topology of the basolateral isoform of the Na-K-2Cl
cotransporter (12). Also shown in Fig. 10 are the
differences between the flounder and the rat cotransporters. flTSC
exhibits three putative N-glycosylation sites, whereas rTSC has only
two of them. The highest degree of identity (~80%) is present along
the membrane-spanning domains. However, in this segment there are more
charged amino acid residues in rTSC than in flTSC. The structural
requirements for ion specificity or affinity in TSC are completely
unknown. However, it has been shown in the basolateral isoform of the
Na-K-2Cl cotransporter that the affinity for the cotransported ions and
the diuretic is determined by the central segment containing the
membrane-spanning domains, and not by the NH2- or
COOH-terminal domains (16). Thus it is possible that the
greater number of charged residues in rTSC could be the reason for the
higher affinity for both ions in the rat cotransporter. The degree of
identity is lower outside the membrane-spanning domains. At the
COOH-terminal domain, the identity is ~55% and, at the
NH2-terminal domain, ~20%. In addition, although there are several putative protein phosphorylation sites shared by both cotransporters in these domains, as Fig. 10 shows, there are some sites
that are present in only one or the other. The different NH2- and COOH-terminal domains between rTSC and flTSC could
be the reason for differences in regulation between these two isoforms, such as the different response to extracellular osmolarity. In conclusion, we have found significant regulatory, kinetic, and pharmacological differences between the flounder and rat isoforms of
the TSC.
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ACKNOWLEDGEMENTS |
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We are grateful to members of the Molecular Physiology Unit for suggestions and stimulating discussions.
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FOOTNOTES |
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This work was supported by research grants 97629m from the Mexican Council of Science and Technology (CONACYT) and 75197-553601 from the Howard Hughes Medical Institute. A. Monroy was supported by scholarship grants from CONACYT. G. Gamba is an International Scholar of the Howard Hughes Medical Institute.
Part of this work was presented at Experimental Biology 2001 (Orlando, FL, March 31 to April 4) and published in abstract form (FASEB J 15: A143, 2001).
Address for reprint requests and other correspondence: G. Gamba, Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico (E-mail: gamba{at}conacyt.mx).
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.
First published November 20, 2001;10.1152/ajprenal.00284.2001
Received 13 September 2001; accepted in final form 14 November 2001.
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