Characterization of the thiazide-sensitive
Na+-Cl
cotransporter: a new model for ions
and diuretics interaction
Adriana
Monroy1,
Consuelo
Plata1,
Steven C.
Hebert2, and
Gerardo
Gamba1
1 Molecular Physiology Unit, Instituto Nacional de la
Nutrición Salvador Zubirán and Instituto de Investigaciones
Biomédicas, National University of Mexico, Tlalpan 14000 Mexico
City, Mexico; and 2 Division of Nephrology and Hypertension,
Department of Medicine, Vanderbilt University Medical Center,
Nashville, Tennessee 37232
 |
ABSTRACT |
The thiazide-sensitive
Na+-Cl
cotransporter (TSC) is the major
pathway for salt reabsorption in the apical membrane of the mammalian
distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both
cotransported ions, with the Michaelis-Menten constant
(Km) for Na+ of 7.6 ± 1.6 mM
and for Cl
of 6.3 ± 1.1 mM, and Hill coefficients
for Na+ and Cl
consistent with
electroneutrality. The affinities of both Na+ and
Cl
were increased by increasing concentration of the
counterion. The IC50 values for thiazides were affected by
both extracellular Na+ and Cl
. The higher the
Na+ or Cl
concentration, the lower the
inhibitory effect of thiazides. Finally, rTSC function is affected by
extracellular osmolarity. We propose a transport model featuring a
random order of binding in which the binding of each ion facilitates
the binding of the counterion. Both ion binding sites alter
thiazide-mediated inhibition of transport, indicating that the
thiazide-binding site is either shared or modified by both
Na+ and Cl
.
metolazone; distal tubule; osmolarity; salt reabsorption
 |
INTRODUCTION |
THE THIAZIDE-SENSITIVE
NA+-Cl
cotransporter is the major NaCl
transport pathway in the apical membrane of the mammalian distal convoluted tubule (DCT). The presence of such a cotransporter was
suggested by Kunau et al. (21), following the observation that chlorothiazide inhibited salt reabsorption in the distal portion
of the nephron. Later micropuncture studies (7,
9) provided strong evidence supporting the existence of a
Na+, Cl
-coupled transport mechanism of salt
reabsorption in the mammalian DCT that was specifically inhibited by
thiazides. After these studies, Beamount et al. (2)
observed that tracer [3H]metolazone was able to bind with
two different binding sites in plasma membranes: one with high affinity
[dissociation constant (Kd) = 4.27 nM]
and the other with low affinity (Kd = 289 nM). The high-affinity [3H]metolazone binding site was
found to be present only in renal cortical membrane preparations, and
not in those from outer or inner renal medulla or other organs; the
binding was selectively blocked by thiazides, with an affinity profile
that was similar to their potency as clinical diuretics. These binding
properties of the high-affinity site for metolazone were considered
compatible with a putative thiazide receptor, that is, with the
thiazide-sensitive Na+-Cl
cotransporter.
[3H]metolazone binding to renal cortical membranes was
subsequently used to study this receptor (3,
6, 30).
The primary structure of the thiazide-sensitive
Na+-Cl
cotransporter [TSC or NCC] has been
elucidated from cloning of cDNAs from the winter flounder urinary
bladder (15) and from rat (rTSC) (14) and
human kidney (hTSC) (29). TSC belongs to the superfamily of electroneutral cation-coupled Cl
cotransporters from
which seven genes have been identified: one encodes for the
thiazide-sensitive Na+-Cl
cotransporter
(14, 15), two genes encode the
bumetanide-sensitive Na+-K+-2Cl
cotransporter (14, 32) and four genes encode
the K+-Cl
cotransporters (16,
23, 25). rTSC mRNA is expressed in renal
cortex and rTSC protein is expressed at the apical membrane of DCT
cells of rat and human kidney (24, 26). In
humans, the TSC gene is localized on chromosome 16 and mutations of
this gene have been linked to Gitelman's syndrome (29),
an autosomal recessive disease featuring chronic arterial hypotension,
hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria.
Targeted disruption of the TSC gene in mice resulted in a partial
Gitelman's-like syndrome (27).
The functional characteristics of TSC from the winter flounder urinary
bladder have been studied in some detail (15), revealing that teleost TSC exhibits Km values for
Na+ and Cl
of 25.0 ± 0.4 and 13.5 ± 0.2 mM, respectively. Hill coefficients for both ions were close to
unity, consistent with electroneutral cotransport. In addition,
thiazide inhibition of flounder TSC function revealed a potency profile
that was similar to that previously shown for inhibition of
Cl
-dependent Na+ absorption in the flounder
urinary bladder [assessed as the short-circuit current
(22)] and for thiazide competition for the high-affinity [3H]metolazone binding site on rat kidney cortical
membranes (2). In this study we present a functional
characterization of the mammalian Na+-Cl
cotransporter, rTSC, as expressed in Xenopus laevis oocytes.
 |
METHODS |
X. laevis oocytes preparation.
Adult female X. laevis frogs were purchased from two
different vendors: Nasco (Fort Atkinson, MI) and Carolina Biological Supply (Burlington, NC). Oocytes were surgically harvested from anesthetized frogs under 0.17% tricaine and incubated during 1 h
under vigorous shaking in 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 B after which oocytes were washed four times in ND-96 and manually defolliculated. Oocytes were incubated overnight in ND-96 at 18°C supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml of gentamicin. Stage V-VI oocytes (8) were then injected with 50 nl of water or rTSC cRNA at a concentration of 0.5 µg/µl or 25 ng cRNA per oocyte. After injection, oocytes were incubated 3-4 days in ND-96 with sodium pyruvate and gentamicin. The incubation medium was changed every 24 h. The day before the uptake experiments were performed, oocytes were switched to a Cl
-free ND-96 [(in mM) 96 Na+-isethionate, 2 K+-gluconate, 1.8 Ca2+-gluconate, 1.0 Mg2+-gluconate, 5 HEPES, 2.5 sodium pyruvate, 5 mg%
gentamicin, pH 7.4] for at least 12 h (15).
In vitro rTSC cRNA translation.
To prepare the rTSC cRNA, rTSC cDNA in pSPORT1 (14) was
linearized at the 3' end by using Not I from Boehringer
(Mannheim, Germany) and cRNA was transcribed in vitro, by
using the T7 RNA polymerase mMESSAGE kit (Ambion). Transcription
product integrity was confirmed on agarose gels, and concentration was
determined by absorbance reading at 260 nm (DU 640, Beckman, Fullerton,
CA). cRNA was stored frozen in aliquots at
80°C until used.
Assessment of the Na+-Cl
cotransporter
function.
Functional analysis of the Na+-Cl
cotransporter was assessed by measuring tracer
22Na+ uptake (New England Nuclear) in groups of
at least 15 oocytes. 22Na+ uptake was measured
by using the following protocol: 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 60-min uptake
period in a K+-free isotonic medium. For most experiments
the isotonic medium contained (mM) 40 NaCl, 56 N-methyl-D-glucamine (NMDG)-Cl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH7.4, supplemented with 1 mM ouabain, 100 µM
bumetanide, 100 µM amiloride, and 2.5 µCi of
22Na+. Ouabain was added to prevent sodium exit
via the Na+-K+-ATPase, bumetanide to inhibit
the oocyte Na+-K+-2Cl
cotransporter, and amiloride to block other Na+ pathways in
the oocytes, such as the Na+-H+ antiporter and
Na+ channels.
To determine the ion transport kinetics and the order of ion binding to
the Na+-Cl
cotransporter, we performed
experiments using varying concentrations of Na+ and
Cl
. To maintain osmolality and ionic strength, gluconate
was used as a Cl
substitute and NMDG as a Na+
substitute. The sensitivity and kinetics for thiazide-type diuretics were assessed by exposing groups of rTSC cRNA-injected oocytes to each
diuretic at concentrations varying from 10
9 to 10
4
M. For these experiments, the desired concentration of the
diuretic was present in both the incubation and uptake periods. In
addition, concentration-dependent effect of thiazides on rTSC function
was assessed by using uptake solutions containing different
concentrations of extracellular Na+ or Cl
.
Finally, uptake experiments were also performed by using three different osmolarity conditions for the oocytes: hypotonicity (110 mosmol/kgH2O), isotonicity (210 mosmol/kgH2O),
and hypertonicity (305 mosmol/kgH2O). All solutions
for these experiments contained 40 mM NaCl, which resulted in an
osmolarity of ~110. For the isotonic and hypertonic solutions,
osmolarity was adjusted by adding 90 mM or 190 mM sucrose, respectively.
All uptakes were performed at 30°C. At the end of the uptake period,
oocytes were washed 5 times in ice-cold uptake solution without isotope
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.
Statistical significance is 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 by using Bonferroni correction or by the
Kruskal-Wallis one-way analysis of variance on ranks with Dunn's
method for multiple comparison procedures, as needed.
 |
RESULTS |
Expression of rTSC in X. laevis oocytes.
We have previously shown that X. laevis oocytes do not
exhibit endogenous expression of the thiazide-sensitive
Na+-Cl
cotransporter (14,
15). Figure 1 shows a
summary from six experiments of rTSC expression in oocytes.
Na+ uptake increased from a level of 157 ± 10 pmol · oocyte
1 · h
1 in
water-injected oocytes to a value of 7,555 ± 228 pmol · oocyte
1 · h
1 in rTSC
cRNA-injected oocytes. Thus injection of X. laevis oocytes with rTSC cRNA resulted in an average 48-fold increase in
Na+ uptake (range 40 to 150-fold in different experiments).
The increased Na+ uptake was chloride dependent and
thiazide sensitive, with uptake in the absence of extracellular
Cl
being 709 ± 114 pmol · oocyte
1 · h
1 and in the presence of
10
4 M metolazone being 1,039 ± 118 pmol · oocyte
1 · h
1. Control uptake in
water-injected oocytes collected from different frogs varied from
70 ± 24 to 450 ± 34 pmol · oocyte
1 · h
1 but was always
insensitive to thiazides.

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Fig. 1.
Functional expression of rat thiazide-sensitive
Na+-Cl cotransporter (rTSC) in Xenopus
laevis oocytes that were injected with water or with 25 ng of cRNA
from rTSC, as indicated. In rTSC-injected oocytes, Na+
uptakes were assessed in presence of Na+ and
Cl (control), in absence of extracellular
Cl or in presence of 10 4 metolazone (MTZ),
as indicated. Each bar represents a mean of 140 oocytes extracted from
6 different frogs. 22Na+ uptake was performed
during 60 min. *Significantly different from uptake in control group
(P < 0.001).
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Figure 2 shows that Na+
uptake in water-injected oocytes was small and linear during 30 min of
uptake. In rTSC cRNA-injected oocytes, the uptake increased rapidly and
was also linear during the first 30 min. Thus we used a 60-min uptake
period for all experiments, except when evaluating ion kinetic analyses
where 15-min uptakes were performed.

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Fig. 2.
Time course over 30 min of Na+ uptake in
X. laevis oocytes injected with water ( ) or with
rTSC cRNA ( ). Each circle represents the mean ± SE of 15 oocytes. Uptake at 30 min in rTSC-injected oocytes was
thiazide sensitive (data not shown).
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Kinetics of ion binding in rTSC.
Figure 3 shows the Na+
dependency (Fig. 3A) and Cl
dependency (Fig.
3B) of Na+ uptake in rTSC cRNA-injected oocytes.
Uptakes were performed with a fixed concentration of Na+ or
Cl
at 40 mM, with changing concentrations of the
counterion from 0 to 40 mM. Uptakes were also measured in
water-injected oocytes (data not shown) and the mean values for water
groups were subtracted from corresponding rTSC groups to assess only
the 22Na+ uptake due to rTSC. As shown in Fig.
1, Na+ uptake in H2O-injected oocytes is low,
making this latter correction small. Na+ uptake increased
as the concentration of each transported ion was raised until a plateau
phase was reached at ion concentrations greater than 20-40 mM,
compatible with Michaelis-Menten behavior. The calculated
Km and maximal velocity
(Vmax) for extracellular Na+
concentration were 7.29 ± 2.1 mM and 3,574 ± 349 pmol
· oocyte
1 · h
1, respectively. The
calculated apparent Km and
Vmax values for extracellular Cl
concentration were 6.48 ± 1.54 mM and 3,542 ± 286 pmol
· oocyte
1 · h
1, respectively. The
Hill coefficient for both ions remained close to unity: 1.04 ± 0.17 and 1.07 ± 0.14 for Na+ and Cl
,
respectively.

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Fig. 3.
Kinetic analysis of Na+ uptake in
oocytes injected with rTSC cRNA. A: Na+
dependency of Na+ uptake. B: Cl
dependency of 22Na+ uptake. Uptakes were
performed during 15 min with a fixed concentration of Na+
or Cl at 40 mM, with changing concentrations of
counterion from 0 to 40 mM. Uptakes were also measured in
water-injected oocytes (data not shown), and mean values for water
groups were subtracted in corresponding rTSC groups to analyze only
Na+ uptake due to rTSC. As shown in Fig. 1, Na+
uptake in H2O-injected oocytes is low, making this latter
correction small. Lines were fit using Michaelis-Menten equation. The
Hill coefficient for Na+ and Cl was close to
unity: 1.04 ± 0.16 and 1.07 ± 0.14 for Na+ and
Cl , respectively. [Na+]e and
[Cl ]e, extracellular Na+ and
Cl concentrations, respectively.
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To test whether extracellular Na+ and Cl
concentrations influence the binding of the counterion, we examined the
counterion concentration dependency of Na+ uptakes at
varying fixed concentrations of extracellular Na+ or
Cl
. Tables 1 and
2 show the results of these experiments
as the kinetic parameters (Km and
Vmax) for Cl
or Na+,
respectively. Table 1 shows that the apparent Km
for Cl
was significantly affected by extracellular
Na+ concentration. The apparent Km
for Cl
increased from 6.46 ± 1.7 to 21.2 ± 0.4 mM (P < 0.01) when extracellular Na+
decreased from 40 to 2 mM. Thus the higher the sodium concentration, the higher the affinity of rTSC for extracellular Cl
.
Similarly, the Km for Na+ also was
affected by extracellular Cl
. As Table 2 shows,
Km for extracellular Na+ varied from
7.26 ± 2.4 to 41.9 ± 6.9 mM (P < 0.01),
when extracellular Cl
concentration varied from 40 to 2 mM. Thus the higher the chloride concentration, the higher the affinity
of rTSC for Na+.
Kinetics of thiazide inhibition of rTSC.
Thiazide-induced inhibition of rTSC has been considered for years as
the hallmark of the Na+-Cl
cotransporter that
is expressed in the apical membrane of the mammalian DCT and the
teleost urinary bladder. Thus we analyzed the inhibitory kinetics of
several thiazide-type diuretics on rTSC cRNA injected-oocytes. The
results of this series of experiments are shown in Fig.
4. The rank of order for rTSC inhibition
was polythiazide > metolazone = bendroflumethiazide > trichloromethiazide > hydrochlorothiazide > chlorthalidone
(chlorthalidone not shown). rTSC function was not affected by the
addition of a nondiuretic thiazide derivative such as diazoxide, tested
in concentrations from 10
14 to 10
4 M in the
uptake medium (data not shown). In addition, rTSC function was not
inhibited by furosemide or acetazolamide (data not shown).

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Fig. 4.
Kinetic analyses of inhibition of rTSC function by
thiazide-type diuretics. All Na+ uptakes were preformed
during 60 min with thiazides tested at concentrations from
10 8 to 10 4 M. The profile of inhibition was
polythiazide ( ), > metolazone ( ) = bendroflumethiazide ( ) > trichloromethiazide
( ) > hydrochlorothiazide ( ).
Uptakes were performed during 60 min in uptake solution containing 40 mM Na+ and 96 mM Cl .
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Effect of pH on rTSC function and thiazide inhibition.
Table 3 shows that there is no effect of
extracellular pH in the range of 6.0 to 8.0 on both rTSC function and
thiazide sensitivity. Uptakes were performed in solutions containing 40 mM NaCl, with pH of 6.0, 6.5, 7.0, 7.5, and 8.0. As Table 3 shows, all
groups exhibited similar uptakes. In addition, we tested two different thiazides: metolazone at a concentration of 5 × 10
6
M that is just above IC50 and bendroflumethiazide at 5 × 10
7 M that is just below IC50. In these
experiments, pH had no effect on metolazone or bendroflumethiazide rTSC
inhibition of Na+ uptake (not significant by using one-way
ANOVA).
Effects of extracellular ions on thiazide inhibition of rTSC.
Tran et al. (30) observed that binding of tracer
[3H]metolazone to its putative receptor was inhibited by
increased Cl
and stimulated by increased Na+
concentrations. They proposed that thiazides and Cl
competed for the same site or at least for part of the same binding site on the protein. To examine this issue at a functional level, we
evaluated the effect of extracellular Cl
on the kinetics
of inhibition of several thiazides. To this end, we assessed kinetics
of rTSC inhibition of five different thiazide-type diuretics, in the
presence of 2 or 100 mM extracellular Cl
. We used 2 mM
Cl
because this concentration is clearly below the
apparent Km for Cl
(Figs. 3 and
4). The results of these series of experiments are shown in Fig.
5 and Table
4. It is clear that the affinity of rTSC
for each thiazide is shifted to the left in the presence of a lower
extracellular Cl
concentration, indicating that
Cl
affects the binding of thiazide diuretics to rTSC. We
also assessed the effect of extracellular Na+ on inhibition
of rTSC by metolazone. As illustrated in Fig.
6, the IC50 was shifted to
the left as Na+ concentration decreased in the uptake
medium. When Na+ concentration was 2 mM, the
IC50 was 3 × 10
7 M, whereas in the
presence of 100 mM Na+, the IC50 was 2 × 10
6 M. Thus the concentration of Na+ in
extracellular fluid also influences the inhibition of rTSC function by
metolazone.

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Fig. 5.
Kinetics of metolazone, polythiazide,
bendroflumethiazide, and hydrochlorothiazide inhibition of rTSC
function in oocytes in presence of extracellular Cl at
concentration of 100 mM ( ) or 2 mM ( ). rTSC
function is expressed as % of control 22Na+
uptake (60 min) in absence of inhibitor. For these experiments
Na+ concentration in both solutions was 40 mM. The
osmolarity and ionic strength for both high and
low-Cl solutions were similar. *P < 0.05 vs. % rTSC function by using same thiazide concentration in 2 mM
Cl .
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Fig. 6.
Kinetics of metolazone inhibition of rTSC function in
oocytes in presence of extracellular Na+ at concentrations
of 100 mM ( ) or 2 mM ( ). rTSC function is
expressed as % of control 22Na+ uptake (60 min) in absence of inhibitor. For these experiments Cl
concentration in three solutions was 100 mM and NMDG substituted for
Na+ where needed. Thus osmolarity and ion strength for the
three solutions were similar. *P < 0.05 vs. %rTSC
function using same thiazide concentration in 2 mM Na+.
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To further examine the effect of ions on thiazide inhibition of rTSC
function, we assessed the effect of increased concentrations of
extracellular Na+ or Cl
on the inhibitory
effect of metolazone at a concentration of 5 × 10
7
M, the IC50 of this diuretic. For these experiments, all
solutions had the same osmolarity (~210 mosmol/kgH2O), as
well as ionic strength. As Fig. 7 shows,
there is a significant negative correlation between both extracellular
Na+ (r2 = 0.82, P < 0.0001) or Cl
(r2 = 0.80, P < 0.001) and
the percentage of rTSC inhibition by metolazone.

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Fig. 7.
Correlations between extracellular Na+
( and dashed line) or Cl ( and
continuous line) concentration and % of rTSC inhibition by 5 × 10 7 M concentration of metolazone. Concentrations of
counterions were held constant at 80 mM. The correlations were
significant (P < 0.001).
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Regulation of rTSC by osmolarity.
The Na-Cl cotransporter, rTSC, is highly expressed in the apical
membranes of the DCT (24, 26). In this nephron segment, the tubular fluid arriving from the medullary thick ascending limb can
vary in osmolarity from hypotonic to isotonic. Accordingly, we studied
the effect of osmolarity on the transport function of rTSC. The
Cl
-dependent fraction and the thiazide-sensitive fraction
of Na+ uptake was assessed in rTSC-injected oocytes that
were exposed to an uptake medium containing 40 mM NaCl at three
different osmolarities: hypotonic (~110 mosmol/kgH2O),
the osmolarity obtained by the 40 mM NaCl concentration in the uptake
medium; and isotonic (~205 mosmol/kgH2O) or hypertonic
(~310 mosmol/kgH2O) by adding sucrose to the 40 mM NaCl
uptake medium. Thus uptakes were performed in different osmolar
conditions, without changing the extracellular NaCl concentration or
ionic strength. Figure 8 shows a
representative experiment. Compared with the amount of
Cl
-dependent or thiazide-sensitive Na+ uptake
that was observed in hypotonicity, rTSC function was increased by
isotonicity. No further activation was seen with the hypertonic uptake
medium.

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Fig. 8.
Effect of osmolarity in rTSC functional expression.
A: Cl -dependent fraction of Na+
uptake. B: thiazide-sensitive fraction of Na+
uptake in oocytes exposed to uptake media with osmolarities of 110 mosmol/kgH2O (open bars), 210 mosmol/kgH2O
(filled bars), and 310 mosmol/kgH2O (gray bars). Uptakes in
all groups were performed during 60 min. *P < 0.05 vs.
uptake in isotonicity.
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DISCUSSION |
In the present study we have functionally characterized the rat
thiazide-sensitive Na+-Cl
cotransporter,
rTSC. As shown previously (14), rTSC gives rise to
thiazide-sensitive Na+-Cl
cotransport when
expressed in X. laevis oocytes. The kinetic analyses for
both ions reveal that rTSC exhibits very high affinities for
Na+ and Cl
. Hill coefficients for each ion
were unity, consistent with a stoichiometry of 1Na:1Cl and the
electroneutral nature of the cotransport process. The
Km values for both ions were < 8 mM. These
ion affinities are somewhat higher than those obtained for the Na-Cl
cotransporter from winter flounder urinary bladder
[Km for Na+ and Cl
were ~25 and 13 mM, respectively (15)]. The large
central hydrophobic domain containing the 12 transmembrane segments of
the related Na-K-2Cl cotransporter has been shown to determine the
diuretic [in this case bumetanide] and ion binding (20).
Because rat and flounder TSC exhibit ~80% amino acid identity in
this central domain, it seems likely that small changes in TSC
sequences in this region account for the kinetic differences between
these two TSC proteins.
In the DCT, urinary fluid arrives from the loop of Henle with
NaCl concentrations and osmolarities that are usually significantly lower than in plasma, due to the intense reabsorption of ions, without
water, in the thick ascending limb. Thus, to maintain an appropriate
rate of salt reabsorption, the apically expressed Na-Cl cotransporter
in the DCT must have very high affinities for both cotransported ions.
The high Na+ and Cl
affinities of ~8
mM for rTSC are consistent with this model and with the previous
observations by Velázquez and coworkers (31) using
in vivo microperfusion experiments in rat DCT in which they found
half-maximal stimulation of salt transport at Na+ or
Cl
concentrations of ~10 mM. In addition, any reduction
of ion reabsorption in the thick ascending limb or increased flow out
of the loop of Henle will result in increased salt (and osmolarity)
delivery to DCT. In this latter circumstance, the rate of NaCl
transport has been shown to be directly related to the rate of NaCl
delivery to the DCT. In this regard, we found in the present study that NaCl transport by TSC was regulated by osmolarity, independently of
changes in NaCl concentration or ionic strength, at least in oocytes.
Thus it is possible that osmotic-induced activation of TSC could be one
of the mechanisms that account for the known immediate increase in NaCl
reabsorption rate in DCT when a loop diuretic is administered
(4).
Our functional study confirms the predictions made by Tran et al.
(30) using [3H]metolazone binding analysis
to renal cortical membranes and by Chang and Fujita (5)
using a recently developed computer-based program that TSC possesses
two binding sites: one selective for Na+ and another for
Cl
. However, in contrast to their findings, we observed
that the affinity of the cotransporter for Na+ or
Cl
is clearly affected by the concentration of the
counterion in the uptake medium. The Km for
Na+ is affected by Cl
and the apparent
Km for Cl
is affected by
extracellular Na+. The higher the counterion concentration,
the higher the Na+ or Cl
affinity of the
cotransporter. We suggest that these results indicate that the order of
binding for Na+ and Cl
to the cotransporter
is random. As shown in the APPENDIX, to analyze the order
of binding of both ions to the cotransporter we followed the "rapid
equilibrium" approach suggested by Segel (28). According to this model, in random bireactant systems, when
= 1, one ion has no effect on the binding of the other and the apparent
Km (Kapp) is held
constant as the counterion concentration increases; when
> 1 the binding of one ion decreases the affinity for the second ion and
the Kapp increases as the concentration of
counterion increases, and when
< 1 the binding of one ion
increases the affinity of the cotransporter for the counterion, and the
Kapp for the varied ion decreases as the
concentration of the fixed ion increases. Thus the observed mutual
effects of Na+ and Cl
concentration on
Kapp and Vmax in our
results are characteristic for random binding of these ions with an
< 1. If the ion binding were ordered, with Na+
binding first, then we would expect that the Vmax
for chloride remains unaffected. As Table 1 and 2 show, the
Vmax for both ions was affected by the
concentration of the counterion.
The binding of the thiazide-like compound [3H]metolazone
exclusively to cellular membranes from renal cortex has been used for
years as a surrogate to study changes in cotransporter expression with
modulation of physiological conditions and during different pathophysiological states (1, 3,
6, 11-13). Our results show
that TSC function is inhibited by several different thiazides with an
inhibitory profile similar to their potency in clinical medicine, as
well as their potency to block the [3H]metolazone binding
to renal cortical membranes (2). In addition, the
benzothiadiazine derivative and vasodilator drug diazoxide, which
causes vasodilation but does not cause diuresis, possesses no
inhibitory effect on TSC function.
Our data show that the concentration of Na+ as well as
Cl
in the extracellular medium affects the affinity of
rTSC for thiazides. The higher the concentration of both ions, the
lower the thiazide-induced inhibition of rTSC function. For example,
the IC50 for metolazone inhibition of rTSC was shifted by
one order of magnitude to the left when either Cl
(Fig.
5) or Na+ (Fig. 6) in the extracellular uptake medium was
decreased from 100 to 2 mM. As shown in Fig. 7, the higher the
Na+ or Cl
concentration, the lower the rTSC
inhibition by thiazides. On the one hand, our findings are in agreement
with the observations of Tran and co-workers (30), who
found an inhibitory effect of Cl
on thiazide binding to
renal cortical membranes, but otherwise diverge from their finding of a
stimulatory effect of Na+ on thiazide binding. We show that
both Na+ and Cl
decrease the inhibitory
potency of thiazides on TSC, whereas Tran and co-workers found that
raising the Na+ concentration increases the binding of
thiazides to plasma membranes from renal cortex (5,
30). Thus, regarding the Na+ and thiazide
interactions, our result appears to be inconsistent with those of Tran
et al. (30).
Differences between predictions based on tracer diuretic binding to
plasma membranes and analysis of the cotransporter function have been
shown to occur. It was predicted by studies using
[3H]bumetanide binding to renal outer medulla membranes
that, in the Na+-K+-2Cl
cotransporter, bumetanide binds to the second Cl
site
(17). However, recent studies of the
Na+-K+-2Cl
cotransporters using
chimeras and point mutations demonstrate that altering the second
transmembrane domain affects Na+, K+, and
bumetanide kinetics, but not Cl
kinetics
(18-20). If bumetanide and Cl
bind to the same site on the protein, the kinetics of both should be
affected by the same alterations in amino acid sequence. Thus bumetanide binding to the Na-K-2Cl cotransporter appears to be distinct
from the Cl
-binding site.
Finally, on the basis of our data, we propose a modification of
the presently accepted TSC model for NaCl transport and inhibition by
thiazides. In this revised Na-Cl cotransporter model, either ion can
first (and presumably randomly) bind to the transporter, but the
binding of this first ion affects the binding affinity of the second
ion (or counterion). In other words, the occupancy of either ion
binding site increases the probability for occupancy of the other one.
Moreover, both ion binding sites alter thiazide-mediated inhibition of
transport, indicating that the thiazide binding site is either shared
or modified by both Na+ and Cl
.
 |
APPENDIX |
To determine the order of ion binding to the thiazide-sensitive
sodium-chloride cotransporter we used the rapid equilibrium approach by
Segel (28). For a random binding assumption we have
where T is transporter, v is the velocity,
KNa+ and
KCl
are the dissociation constants for Na+ and Cl
, respectively, and
is the
factor by which the dissociation constant of one ion is modified by the
binding of the other ion to the transporter.
Vmax is defined as the product of the rate constant, Kp, and the sum of all states of the
transport molecule.
If
< 1 (the binding of one ion increases the affinity of the
cotransporter for the counterion), the Kapp for
the varied ion decreases as the concentration of the fixed ion increases.
If
= 1 (one ion has no effect on the binding of the other),
the Kapp is equal to the K for the
ion and there are not changes with the variation of the counterion.
If
> 1 (the binding of one ion decreases the affinity for the
second ion), in this case the apparent K value for the
varied ion increases as the concentration of fixed ion increases.
The rapid equilibrium approach for an ordered binding if
Na+ binds first yields
The apparent K for Cl
varies with
varying concentrations of Na+ and the
Vmax remains unaffected by the sodium concentration.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Rosario Muñoz-Clares for her help in
kinetic analysis, Jesús López for his help with frogs'
care, and to members of the Molecular Physiology Unit for their
suggestions and stimulating discussion.
 |
FOOTNOTES |
This work was supported by research Grants 97629m from the Mexican
Council of Science and Technology (CONACYT), 75197-553601 from the
Howard Hughes Medical Institute, and National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-38603 (to G. Gamba) and
DK-38603 (to S. C. Hebert). A. Monroy and C. Plata were supported
by scholarship grants from CONACYT and from the Dirección General
del Personal Académico of the National University of Mexico. G. Gamba is an International Scholar of the Howard Hughes Medical Institute.
Address for reprint requests and other correspondence:
G. Gamba, Molecular Physiology Unit, Instituto Nacional de la
Nutrición Salvador Zubirán and Instituto de Investigaciones
Biomédicas, Natl. Univ. of Mexico, Vasco de Quiroga No. 15, Tlalpan 14000, México City, Mexico (E-mail:
gamba{at}mailer.main.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. §1734 solely to indicate this fact.
Received 12 October 1999; accepted in final form 25 February 2000.
 |
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