Naⴙ/Iⴚ symporter (NIS) mediates electroneutral active
transport of the environmental pollutant perchlorate
Orsolya Dohán*†, Carla Portulano*, Cécile Basquin*, Andrea Reyna-Neyra‡, L. Mario Amzel§, and Nancy Carrasco*¶
Departments of *Molecular Pharmacology and ‡Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461; and §Department of Biophysics
and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205
Edited by H. Ronald Kaback, University of California, Los Angeles, CA, and approved October 30, 2007 (received for review July 31, 2007)
The Naⴙ/Iⴚ symporter (NIS) is a key plasma membrane protein that
mediates active Iⴚ uptake in the thyroid, lactating breast, and other
tissues with an electrogenic stoichiometry of 2 Naⴙ per Iⴚ. In the
thyroid, NIS-mediated Iⴚ uptake is the first step in the biosynthesis
of the iodine-containing thyroid hormones, which are essential
early in life for proper CNS development. In the lactating breast,
NIS mediates the translocation of Iⴚ to the milk, thus supplying this
essential anion to the nursing newborn. Perchlorate (ClO4ⴚ) is a
well known competitive inhibitor of NIS. Exposure to food and
water contaminated with ClO4ⴚ is common in the U.S. population,
and the public health impact of such exposure is currently being
debated. To date, it is still uncertain whether ClO4ⴚ is a NIS blocker
or a transported substrate of NIS. Here we show in vitro and in vivo
that NIS actively transports ClO4ⴚ, including ClO4ⴚ translocation to
the milk. A simple mathematical fluxes model accurately predicts
the effect of ClO4ⴚ transport on the rate and extent of Iⴚ accumu-
lation. Strikingly, the Naⴙ/ ClO4ⴚ transport stoichiometry is elec-
troneutral, uncovering that NIS translocates different substrates
with different stoichiometries. That NIS actively concentrates
ClO4ⴚ in maternal milk suggests that exposure of newborns to high
levels of ClO4ⴚ may pose a greater health risk than previously
acknowledged because ClO4ⴚ would thus directly inhibit the new-
borns’ thyroidal Iⴚ uptake.
stoichiometry
he Na⫹/I⫺ symporter (NIS) is the plasma membrane protein
T that mediates active I⫺ uptake in the thyroid, lactating
breast, and other tissues (1). In all tissues and cells where it is
functionally expressed, NIS couples the inward downhill trans-
location of Na⫹ to the inward uphill translocation of I⫺, estab-
lishing ⬎40-fold I⫺ concentration gradient under steady-state
conditions. Using the inwardly directed Na⫹ gradient generated
by the Na⫹/K⫹ ATPase as its driving force, NIS transports I⫺
with an electrogenic stoichiometry of 2 Na⫹ per I⫺ (2–4). In the
thyroid, NIS-mediated I⫺ uptake is the first step in the biosyn-
thesis of the iodine-containing thyroid hormones triiodothyro-
nine (T3) and tetraiodothyronine (T4), which in turn are major
regulators of intermediary metabolism in virtually the entire
organism. T3 and T4 also are essential early in life for proper
development of the CNS, skeletal muscle, and lungs (3–6). The
main regulator of thyroid NIS activity and overall thyroid
function is thyroid-stimulating hormone (TSH). In the lactating
breast, NIS mediates the translocation of I⫺ to the milk, thus
supplying the anion to the nursing newborn for thyroid hormone
biosynthesis (1, 7).
NIS has long been the basis for diagnostic scintigraphic
imaging of thyroid disease and for the highly effective treatment
of metastatic thyroid cancer with radioiodide. However, no
molecular information on NIS was available until 1996, when we
isolated the cDNA-encoding rat NIS (8). The current secondary
structure model for NIS, based on extensive experimental test-
ing, proposes 13 transmembrane segments with the NH2 termi-
nus facing extracellularly and the COOH terminus intracellularly
(9). NIS activity is blocked by the well known classic competitive
inhibitor, perchlorate (ClO⫺ 4 ), which can cause hypothyroidism
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707207104
and goiter and was once used in the treatment of hyperthyroid-
ism. ClO⫺ 4 has long been of central significance in thyroid
pathophysiology and NIS research. Indeed, ClO⫺ 4 inhibition is
one of the clearest hallmarks of NIS-mediated I⫺ transport in
both the thyroid and other tissues. Mechanistically, however, to
date it has remained uncertain whether ClO⫺ 4 is an NIS blocker
or a transported substrate of NIS (2, 10–14).
Although ClO⫺ 4 occurs naturally in the environment, it also is
produced industrially in large quantities. Because of its high
oxidizing ability, ClO⫺4 is used as a propellant and an explosive.
Large-scale production of ClO⫺ 4 in the United States began in
1940, after which time it increased dramatically owing to demand
by the military and the aerospace industry. As a result, ClO⫺
BIOCHEMISTRY
4
exposure is widespread in the U.S. population, although expo-
sure levels are estimated to be less than the EPA reference dose
(15). The extent of ClO⫺ 4 exposure has led to an intense ongoing
debate regarding its potential health impact (16–20). Several
studies report no linkage between ClO⫺ 4 exposure and thyroid
function (21–24). Conversely, a recent study revealed that ClO⫺ 4
exposure of women with low I⫺ intake and smoke exposure was
associated with a decrease in serum T4 and an increase in serum
TSH levels (25, 26). We carried out a thorough analysis of how
NIS handles ClO⫺ 4 and showed that NIS actively transports it,
including its translocation to the milk. A simple mathematical
fluxes model accurately predicts the effect of ClO⫺ 4 transport on
the rate and extent of I⫺ accumulation. Strikingly, the Na⫹/ClO⫺ 4
transport stoichiometry is electroneutral, uncovering that NIS
translocates different substrates with different stoichiometries
and raising the possibility that other transporters may have the
same capability. That NIS actively concentrates ClO⫺ 4 in the milk
suggests that exposure to high levels of ClO⫺4 may pose a greater
environmental health risk than previously acknowledged be-
cause ClO⫺ 4 would thus directly inhibit the newborn’s thyroidal
I⫺ uptake.
Results
ClO4ⴚ Is Actively Translocated by NIS in Epithelial Mammary Cells in
Vivo, Resulting in ClO4ⴚ Accumulation in the Milk. Our cloning (8)
and subsequent detailed characterization of NIS (2, 9, 27) have
made it possible to analyze the mechanism by which ClO⫺ 4
inhibits I⫺ uptake. We demonstrated electrophysiologically that
I⫺ transport by NIS is electrogenic with a 2 Na⫹:1 I⫺ stoichi-
Author contributions: O.D. and C.P. contributed equally to this work; O.D., C.P., C.B.,
A.R.-N., L.M.A., and N.C. designed research; O.D., C.P., C.B., A.R.-N., L.M.A., and N.C.
performed research; O.D., C.P., C.B., L.M.A., and N.C. analyzed data; and O.D., C.P., C.B.,
L.M.A., and N.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
†Presentaddress: National Institute of Oncology and Institute of Experimental Medicine,
Hungarian Academy of Sciences, Rath Gyorgy u. 7–9, 1122 Budapest, Hungary.
¶To whom correspondence should be addressed. E-mail: carrasco@aecom.yu.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0707207104/DC1.
© 2008 by The National Academy of Sciences of the USA
PNAS Early Edition 兩 1 of 6

Fig. 1. NIS-mediated ClO4⫺ accumulation in maternal milk inhibits thyroidal
I⫺ uptake in nursing pups. (A and C) 131I⫺ imaging of lactating rats treated with
ClO4⫺ (A) or not treated (C). Thirty-four hours after administration of 1 mCi
131I⫺, 131I⫺ uptake was barely visible in the thyroid of treated rats (A) but was
clearly apparent in the gland (T) of nontreated ones (C). (B and D) Pups from
treated rats displayed neither thyroidal 131I⫺ uptake nor urinary accumulation
in the bladder (Bla) (B), whereas pups from nontreated rats displayed both (D).
A 18-nCi probe (P) was used as a reference in all images. (E and F) Quantifi-
cation of thyroidal 131I⫺ uptake. 131I⫺ accumulation values were obtained (SI
Materials and Methods) from two lactating control rats and nine nursing pups
(blue bars) and from two lactating rats treated with ClO4⫺ and 10 nursing pups
(red bars). (G) Milk from ClO4⫺-treated rats inhibits I⫺ uptake in hNIS-
expressing MDCK cells. Steady-state (45-min) I⫺ uptake assays were performed
with 20 ␮M I⫺ and the indicated dilutions of the milk samples (see Material and
Methods for details). Data are presented as the reciprocal of the inhibition of
I⫺ transport relative to the values obtained with milk dilutions from non-
treated dams. The ClO4⫺ in the milk was calculated by multiplying the slope of
the inverse of I⫺ transport as a function of the milk dilution times the NIS Ki for
ClO4⫺ (1/0.0058 ⫻ 1.5 ⫽ 260 ␮M).
ometry, eliciting a positive inward current (2). Positive currents
also were generated by other substrate anions, including ClO3⫺,
SCN⫺, SeCN⫺, and NO3⫺. However, neither ClO⫺ 4 nor perrhe-
nate (ReO⫺ 4 ) elicited any currents (2). Similar findings with
ClO⫺4 were reported by other authors (12, 13). These results
indicate that ClO⫺ 4 is not transported by NIS (acting only as a NIS
blocker), is transported at such low rates that transport is
electrophysiologically undetectable, or is transported with an
electroneutral stoichiometry, rendering the transport process
electrophysiologically silent. To address these possibilities, we
injected 1 mCi 131I⫺ i.p. into lactating rats. Half of the rats
simultaneously received 16 ␮moles NaClO4. After treatment, the
dams were reunited with their pups. To continue their exposure
to ClO⫺ ⫺
4 , ClO4 -treated dams received drinking water containing
2 mM NaClO4 while being fed a normal I⫺ diet. Animals were
imaged 34 h after 131I⫺ administration. Thyroidal I⫺ uptake in
both dams and pups was markedly lower in the ClO⫺ 4 -treated
animals (Fig. 1 A, B, E, and F, red bars) than in the controls (Fig.
1 C–F, blue bars). To investigate whether ClO⫺ 4 not only inhibits
NIS function, but is also actually translocated by NIS to the milk,
we injected 10 ␮moles NaClO4 in PBS or PBS alone into the tail
2 of 6 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707207104
vein of lactating rats and tested whether milk samples inhibited
NIS-mediated I⫺ transport in Mardin–Darby canine kidney
(MDCK) cells stably expressing exogenous human NIS (hNIS).
To facilitate milk collection, dams were given oxytocin after 5
min; 30 min later, milk was collected. Only milk samples from
animals that received ClO⫺ 4 inhibited I
⫺
transport, and the
inhibition was inversely proportional to the milk dilution used
(Fig. 1G). Samples from control animals (i.e., PBS-treated) had
no effect. By charting the reciprocal of the inhibition of I⫺
transport at each milk dilution, the reciprocal of the slope,
combined with the 1.25 ␮M IC50 value (28), yielded 260 ␮M
ClO⫺ ⫺
4 in the milk. Because the ClO4 in the serum was estimated
to be 40 ␮M at time 0 by using 0.75 kg/liters for the volume of
distribution of ClO⫺ ⫺
4 (29), the ClO4 milk/serum concentration
ratio was ⬎6. This ratio is in the same range as that reported in
cow’s milk (30). In conclusion, ClO⫺ 4 is not a nontransported
blocker and reaches the milk by active transport, not passive
diffusion.
Polarized NIS-Transfected MDCK Cells Exhibit Active Vectorial ClO4ⴚ
Transport. A direct determination of whether NIS actively trans-
ports ClO⫺ 36 ⫺
4 in vitro by using ClO4 is not possible because of the
radioisotope’s low specific activity. Therefore, we instead used
an indirect approach in a previously established polarized in vitro
bicameral model system (Fig. 2A Right) (D.O., P.C., C. Ginter,
and C.N., unpublished data) (31, 32). The model recapitulates a
polarized epithelial monolayer (ML) with its basolateral (BL)
surface facing the blood supply and its apical (Ap) surface in
contact with the lumen. WT rat NIS (rNIS) is targeted to the BL
plasma membrane in all polarized epithelial cells in which it is
expressed endogenously (1, 4), so the vectorial NIS-mediated
translocation of any NIS substrate proceeds from the BL inter-
stitium into the cell. From there, the substrate exits the cell
across the Ap plasma membrane most likely by Cl⫺ channels (33,
34). In our model, an ML of polarized epithelial MDCK cells
stably expressing exogenous NIS separates the lower chamber
(BL) from the upper chamber (Ap). In these cells, targeting of
WT NIS also is largely basolateral (i.e., the same as in epithelial
cells that express NIS endogenously) (35). However, because the
exogenous overexpression of WT NIS in MDCK cells saturates
the BL targeting machinery, a small fraction of NIS molecules
are targeted apically (31, 36). Thus, to prevent this kind of
bidirectional transport, which might complicate the quantitative
analysis, instead of WT NIS, we used an rNIS construct that lacks
the last 43 amino acids and has a Flag tag attached to the
protein’s amino terminus (Flag-NIS-575). This construct offers
the distinct advantage that, when stably transfected into MDCK
cells, it is only targeted apically (31). Our goal was to obtain
exclusive polarized targeting of NIS to either surface. Flag-NIS-
575 exhibits the same kinetic parameters as WT NIS (31). This
model assumes that the facilitated diffusion also exists on the BL
surface (33, 34).
Hence, we used Flag-NIS-575-expressing MDCK cells in the
described setup to analyze the vectorial transport of ClO⫺ 4 . We
added 20 ␮M I⫺ to the Ap chamber and followed the change in
I⫺ in both chambers over time. I⫺ was rapidly transported from
the Ap chamber (Fig. 2 A, solid blue line) to the BL chamber
(dashed blue line), and the generated gradient was maintained
for ⬎6 h. When we simultaneously added I⫺ and ClO⫺ 4 to the Ap
chamber, strikingly, I⫺ also was translocated to the BL chamber,
except that transport occurred after a considerable delay (⬎1 h)
(Fig. 2 A, solid and dashed red lines). This finding suggests that
the ClO⫺ 4 in the Ap chamber had to decrease below the Ki for
ClO⫺ ⫺
4 before Flag-NIS-575 started translocating I . When non-
transfected (NT) MDCK cells were used, no changes in I⫺ were
observed (Fig. 2 A, black dotted line), supporting the notion that
no I⫺ transport occurs in the absence of NIS.
To investigate whether ClO⫺ 4 reaches the opposite chamber
Dohán et al.

A B
C D E ClO⫺ 4 was added to NT-MDCK cells, only dilutions from the
Fig. 2. NIS actively transports ClO4⫺. (A) ClO4⫺ delays I⫺ vectorial transport in
polarized Flag-NIS-575-transfected MDCK cells. (Right) Schematic representa-
tion of the polarized cell monolayer in a bicameral setup. Ap, apical chamber;
BL, basolateral chamber; ML, cell monolayer. White cylinders represent Flag-
NIS-575, which is exclusively targeted apically. First, 20 ␮M I⫺ was added to the
markedly inhibitory (Fig. 2C, solid line), indicating that ClO⫺
was translocated to the BL chamber by NIS. When ClO⫺
4
4 was
added exclusively to the BL chamber, only BL dilutions were
inhibitory (Fig. 2D, solid line) because apically located Flag-
NIS-575 does not transport any substrate from the BL chamber.
Finally, when ClO⫺ 4 was added simultaneously to both chambers
(Fig. 2E), Ap dilutions minimally inhibited I⫺ transport (Fig. 2E,
dotted lines), further confirming that Flag-NIS-575 translocated
ClO⫺ 4 from the Ap chamber to the BL chamber, whereas BL
dilutions were highly inhibitory (Fig. 2E, solid lines). When
chamber to which ClO⫺ 4 was added caused inhibition (data not
shown). The final ClO⫺ 4 was estimated in each chamber from the
Ki,ClO4 and the dilution by using the equation v ⫽ Vmax䡠[I⫺]/
[Km,I䡠(1 ⫹ [ClO⫺ ⫺
4 /Ki,ClO4]) ⫹ [I ], as described in Fig. 2. Although
we cannot formally rule out the remote possibility that I⫺
transport inhibition was caused by a ClO⫺ 4 -related metabolite,
rather than ClO⫺ 4 , all our data taken together, and the fact that
in rats 99.5% of administered ClO⫺ 4 is excreted in the urine
unmodified (29), strongly suggest that NIS mediates active ClO⫺ 4
transport.
A Kinetic Study of NIS-Mediated ReO4ⴚ Transport Uncovers an Elec-
troneutral Naⴙ/ReO4ⴚ Stoichiometry. The kinetic parameters of

BIOCHEMISTRY
Ap chamber, and then its concentration was monitored as a function of time transport processes that are electrogenic can easily be deter-
in both the Ap chamber (solid blue line) and the BL chamber (dashed blue line). mined by electrophysiological methods. In contrast, when trans-
The Ap concentration of I⫺ started to decrease (and the BL to increase)
port is not electrogenic, a kinetic analysis is only possible by
immediately. When 20 ␮M ClO4⫺ was added together with I⫺ to the Ap
chamber, there was a delay in the Ap decrease (solid red line) and in the
direct substrate flux measurements. The NIS substrate ReO⫺ 4 is
concomitant BL increase (dashed red line) in I⫺. The concentration of I⫺ in the a structurally similar anion to ClO⫺ 4 , and it similarly inhibits
Ap chamber of NT-MDCK cells remained constant (dotted black line). (B) NIS-mediated I⫺ transport without eliciting currents in electro-
Schematic representation of NIS-mediated ClO4⫺ translocation bioassay. Ali- physiological experiments (2). Thus, a study of ReO⫺ 4 behavior
quots from the Ap or BL chamber were taken from the bicameral setup with may shed light on that of ClO⫺ 4 . In contrast to the low specific
Flag-NIS-575-transfected or NT-polarized MDCK cells ML, diluted, and added activity of 36ClO⫺4 , the specific activity of
186ReO is high enough
4
to separate plated, nonpolarized WT-hNIS-expressing MDCK cells for I⫺ up- to be directly measurable in transport assays (37). Just like ClO⫺ 4,
take assays. (C–E) Effect of aliquot dilutions on I⫺ uptake. First 20 ␮M ClO4⫺ all ReO⫺ 4 was translocated from the Ap chamber to the BL
alone (without I⫺) was added to the Ap chamber (C), the BL chamber (D), or
chamber by an ML of polarized MDCK cells expressing Flag-
both (E). After 2.5 h, Ap (open circles) or BL aliquots (filled circles) were added
to cells seeded on plastic, and I⫺ uptake assays (initial rates) were carried out.
NIS-575. When I⫺ and ReO⫺ 4 were added simultaneously to the
Only ClO4⫺-containing aliquots inhibited I⫺ uptake. Transport rates as a func- Ap chamber, each substrate delayed the transport of the other
tion of the reciprocal of the aliquot dilutions are shown. Data were fitted by [supporting information (SI) Fig. 5], proving that both I⫺ and
nonlinear least squares with the equation v ⫽ Vmax䡠[I⫺]/[Km,I]䡠(1 ⫹ [ClO4⫺]/ ReO⫺ 4 were translocated by NIS. We then carried out a kinetic
Ki,ClO4) ⫹ [I⫺]. The intercept, representing the maximal rate of I⫺ transport at analysis of NIS-mediated ReO4⫺ transport in nonpolarized
infinite dilution, was fixed to the value measured in the absence of ClO4⫺. A Ki MDCK cells grown on plastic. We previously showed that
for ClO4⫺ (Ki,ClO4) of 1.2 ␮M and a Km for I⫺ (Km,I) of 20 ␮M were used. When 20 whereas the affinity of WT hNIS for ReO⫺ ⫺
4 is higher than for I ,
␮M ClO4⫺ was added to the Ap chamber of Flag-NIS-575-transfected MDCK the Vmax of ReO⫺ transport and the steady-state levels of
4
cells, the calculated ClO4⫺ in the Ap chamber 2.5 h later was 2.14 ⫾ 0.80 ␮M; in
accumulation are significantly lower than those of I⫺ (37). In
the BL chamber, where there was no ClO4⫺ at time 0, ClO4⫺ reached 22.95 ⫾ 5.45
␮M. In contrast, when 20 ␮M ClO4⫺ was added to the Ap chamber of NT-MDCK
addition to reproducing these findings with Flag-NIS-575 (Fig. 3
cells, the BL ClO4⫺ was 0.46 ⫾ 0.06 ␮M (data not shown). When 20 ␮M ClO4⫺ was A and B), we also observed that the Km for Na⫹ in the course of
added to the BL chamber of Flag-NIS-575-transfected MDCK cells, the calcu- ReO⫺ ⫺
4 transport was similar to that in I transport (Fig. 3 C and
lated ClO4⫺ after incubation was 1.59 ⫾ 0.41 ␮M in the Ap chamber and 21.88 ⫾ D). Most significantly, the initial rates of Na⫹-dependent ReO⫺ 4
8.19 ␮M in the BL chamber (D) and 1.59 ⫾ 0.41 in the Ap chamber when ClO4⫺ transport followed a hyperbolic curve consistent with electro-
⫺
was added to NT cells (data not shown). Finally, when 20 ␮M ClO4⫺ was added ⫹
neutral Na /ReO4 transport (Fig. 3D), in contrast to the sig-
to both chambers, the resulting ClO4⫺ was 1.34 ⫾ 0.35 in the Ap chamber and moidal curve characteristic of the electrogenic 2:1 stoichiometry
44.22 ⫾ 6.78 ␮M in the BL chamber (E). of Na⫹/I⫺ cotransport (Fig. 3C). Note that the change in initial
rates when the Na⫹ goes from 25 to 100 mM is 4.4-fold for I⫺,
whereas it is only 1.9-fold for ReO⫺ 4 . These data are consistent
without being retained or metabolized in the cells, we used
with the significantly lower Vmax of ReO⫺ 4 relative to that of I
⫺
Flag-NIS-575-transfected or NT polarized MDCK cells and
transport, which coexists with a higher apparent affinity of NIS
added 20 ␮M ClO⫺ ⫺
4 alone (without I ) to the Ap chamber, the for ReO⫺ ⫺ ⫹
4 than for I . The Na dependence of the transport rate
BL chamber, or both. Knowing that Flag-NIS-575 is exclusively was adjusted by nonlinear least squares to the Hill equation.
expressed apically, we took aliquots from the Ap or BL chamber Vmax, Km, the background transport, and the Hill coefficient (n)
after 2.5 h (Fig. 2 A), diluted them, and added them to WT- were allowed to vary to fit the experimental data. For Flag-WT-
hNIS-expressing MDCK cells grown on plastic plates (Fig. 2B) NIS the n value for the Na⫹ dependence of I⫺ transport was
to assess the effect of the aliquot dilutions on I⫺ transport initial 1.90 ⫾ 0.2, and for Flag-NIS-575 it was 1.81 ⫾ 0.11. In contrast,
rates (Fig. 2 C–E). Clearly, inhibition of I⫺ uptake would indicate n for ReO⫺ 4 transport by WT hNIS was 0.92 ⫾ 0.2, and for
that the aliquot contains ClO⫺ ⫺
4 . When ClO4 was added exclu- Flag-NIS-575 it was 0.98 ⫾ 0.18. These findings agree with the
sively to the Ap chamber, Ap aliquot dilutions from Flag-NIS- previous data on the electroneutrality of Na⫹/ReO⫺ 4 transport.
575 MDCK cells caused only modest inhibition of I⫺ uptake (Fig. Given the similarities between ReO⫺ ⫺
4 and ClO4 , and taking all
2C, dotted line), whereas dilutions from the BL chamber were of the prior results together, we conclude that ClO⫺ 4 also is

Dohán et al. PNAS Early Edition 兩 3 of 6

 was mediated by NIS. Therefore, the fact that neither ClO⫺ 4 nor
A B ReO⫺
40
12 4 produced currents in electrophysiological studies (2–4) is

ReO4- (pmol/ g DNA/ 2 min)

I- (pmol/ g DNA/ 2 min)
10 a result of NIS-mediated electroneutral transport.
30 8
Mathematical Model That Accurately Predicts the Competition Be-
⫺
20
6 tween ClO4ⴚ and Iⴚ. The effect of ClO4 on the kinetics of I⫺
4 transport (i.e., a delay in the onset of I⫺ transport) points to a
10 simple mechanism of ClO⫺ ⫺
4 inhibition: ClO4 is transported by
2 ⫺
NIS using the same site as I , but with a lower Km. To test this
possibility, we modeled the behavior of I⫺ transport in the
20 40 60 80 20 40
presence and absence of ClO⫺ 4 (Fig. 4) by using a set of equations
I- ( M) ReO4- ( M)
that describe the time dependence of the concentration of both
ions in both chambers. The model used assumes that I⫺ and
C D ClO⫺ 4 are both transported by NIS and compete for the same
ReO4- (pmol/ g DNA/ 2 min)
8 anion site. As shown before, I⫺ transport is described by
I- (pmol/ g DNA/ 2 min)

40
Michaelis–Menten kinetics with a Km of ⬇10–30 ␮M (2, 4, 8).
30 6 The same kinetics is assumed for the transport of ClO⫺ 4 by using
1.5 ␮M experimentally determined Ki from inhibition experi-
4
20
ments as the Km for ClO⫺ 4 . This Ki is consistent with previously
10 2 reported values (39). Small non-NIS-mediated backflow fluxes
are allowed in the model to account for the steady-state con-
centrations of the ions. Four parameters, the Vmax for I⫺ and
50 100 150
Na+ (mM)
50 100 150
ClO⫺ 4 and the two leakage backflow rate constants Kbk,I and
Na+ (mM) Kbk,ClO4, respectively, are adjusted to fit the experimental data to
equations 3, 4, 5, and 6 in Fig. 4. In this example, 20 experimental
E I- points were fitted by adjusting the four parameters to yield the
curves shown. The excellent agreement of the calculated curves
Flag-WT-NIS Flag-NIS-575 Flag-WT-NIS Flag-NIS-575
with the experimental data provides additional support for the
( M) 9.7 ± 2.2 12.6 ± 0.9 2.3 ± 0.3 1.6 ± 0.2 proposed mechanism. The parameters obtained show that ClO⫺ 4
is indeed transported by NIS at a significant rate (a Vmax only
35.6 ± 2.5 45.2 ± 1.1 10.4 ± 0.4 7.6 ± 0.2
(pmol/ g DNA/2 min)
three times lower than that of I⫺). Its backflow rate constant is
(Na+) (mM) 41.3 ± 8.3 33.3 ± 6.7 30.0 ± 4.4 24.9 ± 4.1 ⬇10 times lower than that of I⫺. The ClO⫺ 4 in both chambers as
(Na+)
a function of time computed with these parameters also is
38.7 ± 1.8 28.9 ± 1.4 8.9 ± 0.4 9.0 ± 0.2
(pmol/ g DNA/2 min) plotted in Fig. 4 (dashed lines). The rate of disappearance of
ClO⫺ 4 in the Ap chamber is almost linear for the first 75 min
Fig. 3. Kinetic analysis of NIS-mediated ReO4⫺ transport in MDCK cells. (A–D)
(dotted light green line), whereas the concentration remains over
Initial rates (2-min time points) of I⫺ (A and C) or ReO4⫺ (B and D) transport by
Flag-WT-NIS (solid lines) or Flag-NIS-575 (dotted lines) were determined at the
three times Km. During that time, the rate of I⫺ disappearance
indicated concentrations of I⫺ (A), ReO4⫺ (B), or Na⫹ (C and D). For A and B, a remains low, providing a rationale for the origin of the lag period
constant Na⫹ of 140 mM was used; for C and D, 20 ␮M I⫺ and 2 ␮M ReO4⫺ were in I⫺ transport (red circles). Once the ClO⫺ 4 is low enough to
used, respectively. Calculated curves in A and B were generated with the allow I⫺ to compete for binding to the anion site, the rate of I⫺
equations v ⫽ Vmax䡠[I⫺]/(Km ⫹ [I⫺]) and v ⫽ Vmax䡠[ReO4⫺]/(Km ⫹ [ReO4⫺]), respec- transport increases and eventually (⬇110 min) becomes identical
tively, by using Gnuplot software. In C and D, isotonicity was maintained to the rate observed in the absence of ClO⫺ 4 (solid blue squares).
constant with choline chloride. Na⫹-dependent data were analyzed with the Significantly, modeling allowed us to estimate the kinetics of
equation v ⫽ Vmax䡠[Na⫹]n/Km ⫹ [Na⫹]n. Data were fitted by nonlinear least ClO⫺ ⫺
4 transport without having to measure the ClO4 . The
squares by using Gnuplot software. Background levels in the kinetic experi- ⫺
demonstration that the rate of ClO4 transport is comparable to
ments were ⬍2% for I⫺ kinetics, ⬍3% for ReO4⫺ kinetics, ⬍4% in the Na⫹-
dependent I⫺ transport, and ⬍0.5% in the Na⫹-dependent ReO4⫺ transport.
that of I⫺, together with the lack of current in the electrophys-
Shown are representative experiments. (E) Km and Vmax values of I⫺ and ReO4⫺ iological measurements of ClO⫺ 4 transport, indicates that active
transport by Flag-WT-NIS and Flag-NIS-575 corrected by percentage of NIS- ClO⫺ 4 transport by NIS, a process driven by the Na chemical
⫹

expressing cells. Representative experiments are shown. Experimental values gradient, uses a Na ⫹ /anion stoichiometr y [1:1 or 2:2
represent the average of triplicate points ⫾ SD. Kinetic experiments were (Na⫹:ClO⫺ ⫺
4 )] different from that of I (2:1).
performed at least three to four times.
Discussion
We present experimental evidence showing that ClO⫺ 4 is actively
actively translocated by NIS in an electroneutral fashion. We translocated by NIS in epithelial mammary cells in vivo, resulting
previously showed that pertechnetate (99mTcO⫺ 4 ), another NIS in ClO⫺ 4 accumulation in the milk (Fig. 1). Indication of the
substrate with similar geometry to ClO⫺ 4 , is actively transported presence of ClO⫺ 4 in the milk was obtained with a functional
by lactating breast (1). 99mTcO⫺ 4 is widely used in clinical bioassay (i.e., by showing that milk samples from ClO⫺ 4 -treated
medicine to monitor NIS activity by scintigraphic imaging (38). rats inhibited I⫺ uptake in MDCK cells stably expressing exog-
99mTc exists only as a radioisotope. We determined that the
enous NIS). We also demonstrated NIS-mediated active vecto-
kinetics of Na⫹ dependence of NIS-mediated 99mTcO⫺ 4 transport rial ClO⫺4 transport in polarized NIS-expressing MDCK cells by
(SI Fig. 6A) was hyperbolic, suggesting that NIS translocates using a different bioassay in a bicameral setup (Fig. 2), thus
99mTcO⫺ in an electroneutral fashion, just like NIS-mediated
4 overcoming the difficulty of direct determinations of 36ClO⫺ 4
ClO⫺ ⫺
4 and ReO4 transport. Furthermore, we observed a signif- because of this radioisotope’s extremely low specific activity and
icant concentration gradient of 99mTcO⫺ 4 in the milk with respect limited availability of chemical methods, such as IC/MSMS (30,
to the serum in rats in vivo 30 min after administration of 40, 41). MDCK cells stably transfected with Flag-NIS-575, a rat
99mTcO⫺. A 30-fold concentration gradient was reached at the
4 NIS construct that is exclusively expressed apically, translocated
3-h time point. As expected, 99mTcO⫺ 4 translocation to the milk I⫺ from the apical to the basolateral chamber even when I⫺ was
was inhibitable by ClO⫺4 (SI Fig. 6B), indicating that the process added simultaneously with ClO⫺ 4 to the apical chamber. The fact

4 of 6 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707207104 Dohán et al.


Fig. 4. Transport of I⫺ and ClO4⫺ from the Ap chamber to the BL chamber and
mathematical modeling of vectorial flow. The I⫺ was measured in both chambers
at the indicated times. The experiments being modeled used cells transfected
with Flag-NIS-575 (31) that transport I⫺ (and ClO4⫺) from the chamber facing the
Ap surface of the cells to the chamber facing the BL surface. Open symbols, I⫺ in
the BL chamber; filled symbols, I⫺ in the Ap chamber. Lines represent the flows
calculated by using equations 4, 6, 7, and 8. Four adjustable parameters (Vmax,I,
Vmax,ClO4, Kbk,ClO4, and Kbk,I) were used to fit the data. ClO4⫺s (light and dark dashed
lines) in both chambers were not measured; they were calculated by using
equations 8 and 6 after adjusting the parameters to the experimental I⫺ values.
The time dependence of the I⫺ and ClO4⫺ in the BL and Ap chambers was modeled
with a minimal model based on a limited number of assumptions and parameters.
It was assumed that all transport from the Ap chamber to the BL chamber was
mediated by NIS with a rate describable by the Michaelis–Menten equation. The
dependence of the rate on the Na⫹ was not included because all experiments
were carried out at saturating Na⫹. It also was assumed that both I⫺ and ClO4⫺ use
the same NIS-binding site and, therefore, compete with each other for the site in
the transporter with inhibition constants equal to their Km values (KM,I ⫽ 20 ␮M
for I⫺; KM,C ⫽ 1.5 ␮M for ClO4⫺). The time dependence of the concentration of both
ions in the BL and Ap chambers is obtained by integrating equations 1 and 2. A
concentration-dependent backflow is necessary to account for the final steady-
state concentrations. The parameter Kbk is the backflow rate constant. The
subscript t indicates concentrations at time t and 0 initial concentrations (t ⫽ 0).
Numerical integration of these equations was carried out by a specially written
program, in which the concentrations on the right side of equations 3 and 5 were
taken as those at time t, and those on the left side were taken as concentrations
at time t ⫹ ⌬t to derive equations 7 and 8. Four constants (Vmax,I, Vmax,C, Kbk,I, and
Kbk,C) were adjusted manually to minimize the sum of the squares of the differ-
ences between observed and calculated I⫺ values.
Dohán et al.
that I⫺ transport under these conditions occurred only after a
considerable delay indicates that ClO⫺ 4 was translocated first. We
show further that when ClO⫺ 4 alone was added to the apical
chamber, it was transported by NIS into the cells. From there,
without being metabolized, the anion continued through diffu-
sion to the BL chamber (Fig. 2). This finding was demonstrated
by the observation that BL dilutions inhibited I⫺ transport in WT
hNIS-expressing MDCK cells grown on plastic plates, which is an
indication that the dilutions contained ClO⫺ 4.
Because Na⫹-dependent ClO⫺ 4 transport is not electrogenic,
its kinetic analysis could only be carried out by direct substrate
flux measurements. However, as noted previously, such mea-
surements are precluded by the low specific activity of 36ClO⫺ 4.
Thus, we instead analyzed the kinetic parameters of NIS-
mediated transport of a structurally similar anion, ReO⫺ 4 , taking
advantage of the higher specific activity of the radioisotope
186
ReO⫺ 4 . We found that the initial rates of NIS-mediated,
Na⫹-dependent ReO⫺ 4 transport yielded a hyperbolic curve
indicative of an electroneutral stoichiometry, strongly suggesting
that the NIS-mediated Na⫹/ClO⫺ 4 transport stoichiometry also is
electroneutral. This finding is in stark contrast to the electro-
genic 2:1 Na⫹/anion stoichiometry observed with most other
NIS substrate anions, demonstrating that NIS translocates dif-
ferent substrates with different stoichiometries, an unprece-
BIOCHEMISTRY
dented property for any transporter. Indeed, this observation
raises the possibility that other transporters also may have the
same capability. Finally, we show that a simple mathematical
model based on our I⫺ transport data in NIS-transfected polar-
ized MDCK cells in vitro accurately predicts the degree of
competition between ClO⫺ 4 and I .
⫺
This research will undoubtedly impact the debate on the public
health effects of ClO⫺ 4 pollution. Whereas several studies re-
ported no linkage between ClO⫺ 4 exposure and thyroid function
(21–24), recent studies (25, 26) indicated that long-term ClO⫺ 4
exposure, even at lower doses, correlates with decreased T4 and
⫺
increased TSH levels in women with low I intake levels and
tobacco smoke exposure. Our findings make it clear that, aside
from the effects of ClO⫺ 4 on the health of adult women, if a
nursing mother is exposed to high levels of ClO⫺ 4 , the anion will
not only inhibit I⫺ accumulation in the milk, which is critical for
thyroid hormone biosynthesis by the newborn, but will be
actively concentrated in the milk and, thus, directly inhibit the
newborn’s thyroidal I⫺ uptake. This effect would have poten-
tially serious consequences for the child’s mental and physical
development.
Materials and Methods
Iⴚ Transport Assays. Initial rate (2-min) I⫺ uptake assays were
performed as previously described (41). In Na⫹-dependent
initial rate transport assays, 20 ␮M 125I⫺, 2 ␮M 186ReO⫺ 4 , or 5
nCi/␮l of 99mTcO⫺ 4 was incubated with 0–160 mM Na , and
⫹
isotonicity was kept with choline chloride. Steady-state I⫺ up-
take assays were performed in hNIS-MDCK seeded on plastic
with milk samples (diluted in HBSS with 20 ␮M KI and 125I⫺ at
100 mCi/mmol) from ClO⫺ 4 -injected or control animals. For the
NIS-mediated ClO⫺ 4 -translocation bioassay, aliquots from the
Ap or BL chamber were diluted in HBSS containing 20 ␮M 125I⫺
at 50 ␮Ci/␮mol. Data were processed by using the equation v ⫽
Vmax䡠[A⫺]/(Km ⫹ [A⫺]) for I⫺ and ReO⫺ 4 kinetics and v ⫽
Vmax䡠[Na⫹]n/(Km⫹[Na⫹]n) for Na⫹-dependent kinetics. Data
were fitted by nonlinear least squares with Gnuplot software.
Nonspecific background, as measured in NT cells, was sub-
tracted. All parameters were measured at least in triplicate.
Measurement of Vectorial Transport. To assess vectorial I⫺ trans-
port through the polarized epithelial monolayer of NT-MDCK
cells or cells stably transfected with Flag-NIS-575, we added 1 ml
of HBSS containing 20 ␮M I⫺ supplemented with carrier-free
PNAS Early Edition 兩 5 of 6
Na125I at a specific activity of 100 mCi/mmol in the Ap chamber
with or without 20 ␮M NaClO4 and 1 ml of buffered HBSS in
the BL chamber of the filter-grown polarized epithelial mono-
layer. To assess the I⫺-induced delay on ReO⫺ 4 transport and vice
versa, 1 ml of 2 ␮M 186ReO⫺ 4 at a specific activity of 1 mCi/␮mol
in buffered HBSS solution with or without 100 ␮M KI was added
to the Ap chamber or 1 ml of 20 ␮M 125I⫺ at a specific activity
of 100 mCi/mmol with or without 20 ␮M ReO⫺ 4 . Then 10-␮l
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