BBRC

Biochemical and Biophysical Research Communications 323 (2004) 11451150

www.elsevier.com/locate/ybbrc

A role for ZnT-1 in regulating cellular cation inux

Dror Segala, Ehud Ohanaa, Limor Besserb, Michal Hershnkelb,
Arie Morana, Israel Seklera,*
a
b

Department of Physiology, Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel
Department of Morphology, Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel
Received 15 August 2004
Available online 17 September 2004

Abstract
The ZnTs are a growing family of proteins involved in lowering or sequestration of cellular zinc. Using uorescent measurements
of zinc transport we have addressed the mechanism of action of the most ubiquitously expressed member of this family, ZnT-1. This
protein has been shown to lower levels of intracellular zinc though the mechanism has remained elusive. The rate of zinc eux in
HEK293 cells expressing ZnT-1 was not accelerated in comparison to control cells, suggesting that ZnT-1 may be involved in regulating inux rather than eux of zinc. Co-expression of the L-type calcium channel, a major route for zinc inux, and ZnT-1
resulted in a 3-fold reduction in the rate of zinc inux in HEK293 and PC-12 cells, indicating that ZnT-1 modulates zinc permeation
through this channel. Immunoblot analysis indicates that ZnT-1 expression does not modulate LTCC expression. Our ndings
therefore indicate that ZnT-1 modulates the permeation of cations through LTCC, thereby, regulating cation homeostasis through
this pathway. Furthermore, ZnT-1 may play a role in cellular ion homeostasis and thereby confer protection against pathophysiological events linked to cellular Ca2+ or Zn2+ permeation and cell death.
2004 Elsevier Inc. All rights reserved.
Keywords: Zinc transporter; ZnT-1; L-type calcium channel; Zinc eux; Ion homeostasis; Neuronal zinc toxicity

Massive inux of zinc ions, released from glutamatergic synaptic vesicles in the brain, during ischemic
episodes, seizures, and traumatic brain injury or from
b-cells in the pancreatic islets of Langerhans is considered a major factor in subsequent cell death [16]. Zinc
permeates into cells via two major channel types: the
Ca-A/K, a subtype of the glutamate receptors, and voltage sensitive (L-type) calcium channels (LTCC). While
the former channel type is restricted to hippocampalpyramidal neurons [7,8], the latter is expressed in a wide
variety of tissues, and cell types [9,10]. In addition, vectorial transport of zinc is known to be mediated by ZIP,
acting as a HCO3/Zn co-transporter [11].
Calcium permeates cells via the LTCC in virtually
every organ, but is perhaps best known for its role in
*

Corresponding author. Fax: +972 8 6477627.

E-mail address: sekler@bgumail.bgu.ac.il (I. Sekler).

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.08.211

cardiovascular disease, particularly stroke, and hypertension [12]. The high zinc permeability of the LTCC
is also implicated in zinc signaling and toxicity in cardiomyocytes, neurons, glia, and pancreatic b cells [9,1315].
Despite the general importance and the pathophysiological implications of inux via the LTCC, no mechanism
regulating zinc inux through this pathway has been described to date.
The Zn-T family consists, thus far, of 10 members
that are involved in either lowering cytoplasmic zinc
(mediated by ZnT-1) or in zinc sequestration into secretory, and synaptic vesicles (mediated by ZnT-2-7) [16
18]. Among the ZnT family members, ZnT-1 is the most
ubiquitously expressed, and the only member found on
the cellular plasma membrane. ZnT-1 is also the only
member with a well-documented role in conferring resistance against zinc toxicity in multiple tissues and cell
types [13,15,19]. Expression of ZnT-1 is induced by

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D. Segal et al. / Biochemical and Biophysical Research Communications 323 (2004) 11451150

dietary or vesicular zinc by a mechanism involving the

metal transcription factor, MTF [20]. In the brain the
expression of ZnT-1 is developmentally regulated and
recent studies indicate that the expression of ZnT-1 is
correlated with the appearance of chelatable zinc [21].
Knockout of the ZnT-1 gene is lethal during early
embryonic stages, indicating that this protein is essential
for embryonic development [20]. Although the above
studies point toward the physiological and pathophysiological importance of ZnT-1 expression, the functional
mechanism linking ZnT-1 expression to cellular zinc
homeostasis is poorly understood. Of particular interest
is the unresolved question of whether ZnT-1 acts as a
transporter or as a regulator of zinc transport through
known permeation pathways.
Much more is known about calcium homeostasis
than that of zinc. Ironically, many of the tools that were
originally designed for monitoring [Ca2+]i can be used
more eectively for [Zn2+]i determination. Fura-2 for
example, a classical uorescent probe for monitoring
Ca2+, has a 100-fold higher anity for Zn2+ [9,22].
Moreover, changes in intracellular Zn2+ can be easily
calibrated and distinguished from changes in intracellular Ca2+ by the use of zinc ionophores and heavy metal
chelators. The Zn2+ chelator TPEN, for example, has an
approximately 1010-fold higher anity for Zn2+ compared to Ca2+ [23]. We therefore studied the mechanism
linking ZnT-1 expression to reduction of [Zn2+]i using
uorescent ion imaging. In the present study, we report
on the mechanism of ZnT-1 activity; our ndings suggest that ZnT-1 acts as a modulator of the LTCC,
down-regulating not only Zn2+ but also Ca2+ inux
through this pathway and thereby protecting cells from
the eects of excessive cation permeation. The physiological implications for cellular Zn2+ and Ca2+ homeostasis are discussed.

Materials and methods

Cell culture. PC-12 cells (rat pheochromocytoma cell line) and
HEK293 (human embryonic kidney cell line) were grown as described
previously [2426]. Cells were seeded on glass cover slides in a 60 mm
tissue culture plate for 12 days prior to uorescent imaging. Cells
were then transfected as previously described [25] with 3 lg of mouse
ZnT-1 plasmid (kindly provided by Dr. R. Palmiter, University of
Washington, Seattle) or co-transfected with 3 lg ZnT-1, 3 lg of the
a-subunit, and 1.2 lg of the b-subunit of LTCC (kindly provided by
Dr. F. Hofmann, T.U. Munchen, Germany). In some experiments cells
were co-transfected with an EYFP plasmid (Clontech, Palo Alto,
California, USA) serving as a reporter, used at 1/10 ratio. Transfection
of HEK293 cells was performed using calcium phosphate precipitation
[27] while PC-12 cells were transfected with either Fugene-6 (Roche) or
Superlipofectamine (Gibco-BRL) used as directed by the manufacturer. Typically, the transfection eciencies reached 7080% and
4050% for the HEK293 and PC-12 cells, respectively.
Fluorescent imaging. The imaging system consisted of a Zeiss
Axiovert 100, inverted microscope, Polychrome II monochromator
(T.I.L.L Photonics, Germany), and a cooled CCD (PCO). Fluorescent

imaging measurements were acquired using Axon Imaging Workbench

2.2. All results shown are means of 46 independent experiments, with
averaged responses of 3050 cells in each experiment.
Intracellular zinc imaging. Cells were incubated for 30 min, with
5 lM Fura-2 AM (TEFLabs) in 0.1% BSA in sodium Ringers solution. Following dye loading, the cells were washed with Ringers
solution and the coverslips were mounted on a perfusion chamber as
previously described [28]. Fura-2 was excited at 340 and 380 nm
wavelengths and imaged with a 510 nm long pass lter. Intracellular
zinc concentrations were calibrated using the zinc ionophore pyrithione in the presence of 100 lM zinc, followed by the addition of
50 lM TPEN, as previously described [29].
Protein determination and immunoblot. Cells were extracted using
RIPA lysis buer or alternatively using denaturating lysis buer (1%
SDS and 1% Tris base, pH 8.00), both containing a protease inhibitor
mixture (Boehringer Complete protease inhibitor mixture, Roche
Molecular Biochemicals). Protein concentrations were determined
using the modied Lowry procedure [30]. SDS gels and immunoblot
analysis were performed as previously described [31]. ZnT-1 and
LTCC were detected using ZnT-1 antiserum [31] and an antibody
against the cardiac a-subunit of the LTCC (Alomone, Israel) at 1/200
and 1/200 dilution, respectively.

Results
ZnT-1 expression does not alter zinc eux
The ZnT-1 expression level in HEK293 cells was
monitored by immunoblot analysis using the ZnT-1 antibody which stained a polypeptide of 60 KDa (Fig. 1),
corresponding to the expected molecular weight of this
protein. No apparent staining of ZnT-1 was monitored
in vector-transfected cells, attesting to their low endogenous expression of ZnT-1.
Although ZnT-1 has been shown to lower levels of
[Zn2+]i in cells [15,19,32], this lowered accumulation
may reect either an accelerated Zn2+ eux or reduced
Zn2+ inux. We rst addressed the former hypothesis by
monitoring rates of zinc eux in Fura-2 stained
HEK293 cells. The cells were loaded with Zn2+, by perfusing them with sodium-free medium containing zinc,
thereby reversing their endogenous Na+/Zn2+ exchange
[33]. The Fura-2 uorescent signal was then eliminated
by application of the cell permeable Zn2+ chelator,
TPEN (Fig. 2A), indicating that the Fura-2 signal is indeed related to [Zn2+]i. Zinc eux, from Zn2+-loaded

Fig. 1. Expression of ZnT-1 in HEK293 cells. HEK293 cells were

either transfected by CaPO4 with vector alone (control) or with ZnT-1
plasmid. Immunoblot was performed using the indicated amount of
protein.

D. Segal et al. / Biochemical and Biophysical Research Communications 323 (2004) 11451150

1147

Fig. 2. Expression of ZnT-1 in HEK293 cells does not aect zinc accumulation. (A) HEK293 cells loaded with Fura-2 were perfused with Na+-free
Ringers containing 400 lM zinc (see Materials and methods) followed by perfusion with Ca2+- and Zn2+-free Ringers containing TPEN (50 lM).
The reduction in Fura-2 uorescence following TPEN application indicates that the uorescence observed is due to changes in intracellular zinc.
HEK293 cells preloaded with Fura-2 transfected with vector or expressing ZnT-1 were loaded with zinc and then perfused with Ca2+-free Ringers
containing 50 lM zinc (B) or with nominally zinc-free (C) Ringers solution. There was no change in the rate of zinc eux under either condition,
indicating that ZnT-1 does not participate in net zinc eux.

cells, was monitored against an inward Zn2+ gradient of

50 lM extracellular Zn2+ in Ca2+-free Ringers solution
versus 100 nM intracellular Zn2+. As shown in Fig. 2B
expression of ZnT-1 in HEK293 cells did not result in
signicant acceleration of Zn2+ eux against the inward
Zn2+ gradient. The rate of zinc eux in ZnT-1 expressing cells was also not accelerated when cells were perfused with nominally Zn2+-free Ringers (Fig. 2C),
thus eliminating the inward zinc gradient. Taken together, the results of Figs. 1 and 2 indicate that the
expression of ZnT-1 in HEK293 cells does not result
in increased zinc eux.
ZnT-1 regulates zinc permeation through the L-type Ca2+
channel
If ZnT-1 does not catalyze zinc eux, then ZnT-1
may reduce [Zn2+]i instead by lowering the net zinc inux. A primary pathway for zinc permeation into cells
is the L-type Ca2+ channel. However, because endogenous L-type Ca2+ channel activity in HEK293 is relatively low and not well characterized, we utilized two
independent experimental paradigms to determine if,

indeed, ZnT-1 acts by modulating zinc inux through

LTCC. In the rst, the rate of Zn2+ inux was monitored in HEK293 cells heterologously co-expressing
LTCC and ZnT-1. As shown in Fig. 3A, following
opening of the LTCC by depolarization, there was a
3-fold reduction in Zn2+ inux in cells that co-expressed ZnT-1and LTCC. Our second approach was
to monitor Zn2+ inux in PC-12 cells, which express
a well-characterized LTCC. These cells were transfected with the ZnT-1 plasmid [13,34] and Zn2+ inux
was monitored. Cells were depolarized by high-K+
Ringers solution (50 mM KCl replacing NaCl) to open
the LTCC and the rate of Zn2+ inux was determined.
As shown in Figs. 3B and C, transient expression of
ZnT-1 reduced the depolarization-dependent Zn2+ inux in PC-12 cells by 3-fold. The results shown in
Fig. 3 indicate that ZnT-1 lowers intracellular zinc by
reducing Zn2+ inux through the LTCC.
The ZnT-1 dependent reduction of zinc permeation
through the LTCC may have resulted either through
functional interaction of ZnT-1 with the LTCC or modulation of LTCC expression. To address the latter
hypothesis, expression levels of the catalytic a-subunit

Fig. 3. Expression of ZnT-1 in cells reduces zinc inux through LTCC. (A) Rates of depolarization induced zinc inux in HEK293 cells that were
either transfected with the cardiac LTCC isoform or co-transfected with LTCC and ZnT-1. (B) Depolarization induced zinc inux in PC-12 cells that
were either transfected with ZnT-1 or vector alone. (C) Rates of depolarization induced zinc inux in PC-12 cells that were transfected with the ZnT-1.
As seen from the bar graphs the rate of zinc inux in ZnT-1 expressing cells was reduced by 3-fold in both HEK293 and PC-12 cells.

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D. Segal et al. / Biochemical and Biophysical Research Communications 323 (2004) 11451150

12 nM/s in control) indicating that, indeed, ZnT-1 regulates not only Zn2+ but also Ca2+ permeation through
the LTCC.

Discussion
Fig. 4. Expression of ZnT-1 does not alter the expression of LTCC.
(A) Immunoblot of LTCC in HEK293 cells that were either transfected
with ZnT-1 or co-transfected with ZnT-1 and LTCC plasmid. (B)
Immunoblot of LTCC in PC-12 cells that were transfected with ZnT-1
or vector alone. Expression of ZnT-1 in either cell types did not aect
the expression of LTCC.

of LTCC were determined by immunoblotting of PC-12

and HEK293 cell extracts transfected with ZnT-1 (PC12) or co-transfected with ZnT-1 and LTCC
(HEK293). As shown in Fig. 4, expression of LTCC in
PC-12 cells transfected with ZnT-1 versus control was
similar. Furthermore, there was no signicant dierence
in LTCC expression in HEK293 cells that were co-transfected with ZnT-1. Our results thus are consistent with
the hypothesis that ZnT-1 down-regulates zinc permeation by a functional interaction and not by modulating
LTCC expression.
ZnT-1 regulates Ca2+ permeation through LTCC
ZnT-1 regulation of the LTCC aects the main pathway for Zn2+ permeation into cells, though other cations
also permeate via this pathway. Hence, we hypothesized
that ZnT-1 may not only regulate Zn2+ but also Ca2+ inux through this pathway. To address this hypothesis,
we used the same experimental paradigm shown in
Fig. 3 but with the exception that cells were perfused
with Ca2+ Ringers solution instead of Ringers supplemented with Zn2+. Rates of Ca2+ inux were compared
in cells expressing ZnT-1 versus controls. As shown in
Fig. 5, expression of ZnT-1 reduced the rate of Ca2+ inux by 40% (6.8 nM/s in ZnT-1 expressing cells;

ZnT-1 plays a key role in conferring resistance

against toxic concentrations of zinc in multiple cell types
and tissues [35]. Previous studies have suggested [19,32]
that ZnT-1 may lower [Zn2+]i by acting as a zinc extruder in BHK cells. Importantly, however, the Zn2+ extrusion linked to ZnT-1 expression described in these
earlier studies could not be modulated by changing the
concentration of counterions such as K+, Na+, Ca2+
or protons in the experimental solutions nor was it affected by depletion of intracellular ATP. Thus, the
mechanism underlying the reduction of intracellular
Zn2+ by ZnT-1 remained unknown. Our ndings are
consistent with a role for ZnT-1 in lowering intracellular
Zn2+, but suggest that ZnT-1 is acting as a specic regulator of Zn2+ inux through LTCC and not as a Zn2+
pump or transporter per se. This is based on the following ndings: expression of ZnT-1 in PC-12 cells reduced
Zn2+ inux via the LTCC by 3-fold; heterologous co-expression of ZnT-1 and LTCC also reduced Zn2+ inux
in the LTCC expressing cells. This down-regulation of
zinc inux through the LTCC mediated by ZnT-1
changes the balance of zinc inux/eux and thereby
may appear as an increase in the rate of cellular Zn2+ efux. Furthermore, if ZnT-1 had only catalyzed Zn2+ efux, it would have not changed the initial rates of Zn2+
inux mediated by the LTCC as we have shown in
Fig. 3. Indeed, Zn2+ eux mediated by the Na/Zn exchanger [33] lowers intracellular Zn2+ but does not aect
the initial rate of Zn2+ inux mediated by the LTCC in
neurons. The fact that ZnT-1 regulates cation inux via
the LTCC is further supported by our recent demonstration that expression of ZnT-1 strongly attenuates zinc
inux through the LTCC, thereby reducing glial zinc

Fig. 5. Calcium inux is inhibited by the expression of ZnT-1. (A) ZnT-1 was heterologously expressed in PC-12 cells, expressing endogenous LTCC.
The depolarization induced calcium inux was monitored in Fura-2 loaded cells perfused with Ringers solution containing high-K+ (that induced
depolarization) in the presence of calcium (but no zinc). (B) Bar graph showing the rates of calcium inux following depolarization. Rates are
decreased by 2-fold in ZnT-1 expressing cells compared to control cells.

D. Segal et al. / Biochemical and Biophysical Research Communications 323 (2004) 11451150

toxicity [15]. We therefore suggest that the model of

ZnT-1 acting as a regulator of Zn2+ inux is the most
plausible explanation reconciling our studies with previous ones linking ZnT-1 expression and the lowering of
[Zn2+]i.
Transport studies and the use of knockout mice have
demonstrated that the ZnT-2-7 proteins are involved in
accumulation of zinc into intracellular vesicles. For
ZnT-1, however, the functional mechanism linking this
protein to sequestration of zinc is unknown. Because
the LTCC is not a major pathway in these compartments, these transporters (ZnT-2-6) are unlikely to share
the same LTCC-regulating activity demonstrated here
for ZnT-1. Interestingly, the positive inside membrane
potential of these intracellular compartments [36] implies that down-regulation of cationic channels by ZnT
proteins will attenuate zinc eux, in concert with zinc
transporters, can promote zinc accumulation. Studies
on isolated vesicles and zinc imaging of plasma membrane permeabilized cells are underway to determine
the functional mechanisms linking the expression of
these transporters to intravesicular zinc accumulation.
Although ZnT-1 regulates LTCC activity, it does not
aect its expression level, indicating that this regulation
is based on functional interaction between the two proteins. At present, we do not know how ZnT-1 interacts
with the LTCC or whether it acts indirectly to modulate
LTCC activity. Applying an immunoprecipitation approach on HEK293 cells co-expressing ZnT-1 and
LTCC or on PC-12 cells transfected with ZnT-1, using
either ZnT-1 or LTCC antibodies, failed to clearly demonstrate an interaction between the two proteins. This
does not exclude the possibility that the two proteins
do interact. Perhaps, this interaction is not stable enough to withstand the immunoprecipitation procedure
or that the antibodies non-specically interfere with
the LTCC/ZnT-1 interaction sites. Alternatively, ZnT1 may act as a regulator of LTCC by modulating the
activity of signal transduction pathways such as the
PKA or PKC which are known to regulate various
aspects of LTCC activity [37]. Future studies using alternative approaches for probing protein interactions, such
as yeast two hybrid or immunoprecipitation preceded by
chemical crosslinking, may enable us to address this unsolved issue. The possible functional interaction between
these two proteins in hippocampal and cortical pyramidal neurons, where they are both highly expressed, is another area of interest. In these neurons the L-type Ca2+
channels are not only involved in physiological synaptic
transmission, but may also participate in the response to
pathological events, leading to elevated neuronal Zn2+
and Ca2+ during stroke, and epilepsy [3]. By regulating
the expression levels of ZnT-1 and thereby modulating
the activity of the LTCC, synaptic zinc can play a novel
role as a regulator of synaptic transmission. Intriguingly, levels of synaptic zinc and expression of both

1149

ZnT-1 and LTCC are developmentally regulated, pointing to the feasibility of such novel regulatory mechanism
[21,38].
The ubiquitous nature of the LTCC and ZnT-1
expression, and the key role that the former plays in
Ca2+ and Zn2+ homeostasis, underscores the general
importance of this mode of regulation. Furthermore,
this may underlie the lethality seen in ZnT-1 knockout
mice during early embryonic development [20]. Indeed,
our results indicate that ZnT-1 inhibits not only Zn2+
permeation but also Ca2+ inux through the LTCC.
Calcium permeation through this channel mediates arteriole vasoconstriction and subsequent hypertension, and
LTCC blockers are widely used as antihypertensive
agents. It would be interesting, therefore, to study the
role of ZnT-1 in this context. Interestingly, the expression of ZnT-1 in a variety of tissues is upregulated by increased dietary zinc [17,39].

Acknowledgments
We thank Dr. W.F. Silverman for fruitful discussions
and critical reading of the manuscript, and Ms. H. Tamir-Azriel and I. Cohen for technical help with the
immunoblots. This work was partially supported by
the GIF Grant 10588099.01/98 (I.S. and A.M.), ISF
Grant 456/02.1 (I.S. and A.M.), ISF equipment Grant
456/02.2 (I.S.), and BSF Grant 2001101 (M.H.).
References
[1] Y.H. Kim, E.Y. Kim, B.J. Gwag, S. Sohn, J.Y. Koh, Zincinduced cortical neuronal death with features of apoptosis and
necrosis: mediation by free radicals, Neuroscience 89 (1999) 175
182.
[2] J.Y. Koh, S.W. Suh, B.J. Gwag, Y.Y. He, C.Y. Hsu, D.W. Choi,
The role of zinc in selective neuronal death after transient global
cerebral ischemia, Science 272 (1996) 10131016.
[3] D.W. Choi, J.Y. Koh, Zinc and brain injury, Annu. Rev.
Neurosci. 21 (1998) 347375.
[4] C.J. Frederickson, M.D. Hernandez, S.A. Goik, J.D. Morton,
J.F. McGinty, Loss of zinc staining from hippocampal mossy
bers during kainic acid induced seizures: a histouorescence
study, Brain Res. 446 (1988) 383386.
[5] C.J. Frederickson, M.D. Hernandez, J.F. McGinty, Translocation
of zinc may contribute to seizure-induced death of neurons, Brain
Res. 480 (1989) 317321.
[6] S.W. Suh, R.B. Thompson, C.J. Frederickson, Loss of vesicular
zinc and appearance of perikaryal zinc after seizures induced by
pilocarpine, Neuroreport 12 (2001) 15231525.
[7] J.H. Weiss, S.L. Sensi, Ca2+Zn2+ permeable AMPA or kainate
receptors: possible key factors in selective neurodegeneration,
Trends Neurosci. 23 (2000) 365371.
[8] Y. Jia, J.M. Jeng, S.L. Sensi, J.H. Weiss, Zn2+ currents are
mediated by calcium-permeable AMPA/kainate channels in
cultured murine hippocampal neurones, J. Physiol. 543 (2002)
3548.
[9] D. Atar, P.H. Backx, M.M. Appel, W.D. Gao, E. Marban,
Excitationtranscription coupling mediated by zinc inux through

1150

[10]
[11]

[12]

[13]

[14]

[15]

[16]
[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

D. Segal et al. / Biochemical and Biophysical Research Communications 323 (2004) 11451150

voltage-dependent calcium channels, J. Biol. Chem. 270 (1995)

24732477.
W.A. Catterall, Structure and regulation of voltage-gated Ca2+
channels, Annu. Rev. Cell. Dev. Biol. 16 (2000) 521555.
L.A. Gaither, D.J. Eide, The human ZIP1 transporter mediates
zinc uptake in human K562 erythroleukemia cells, J. Biol. Chem.
276 (2001) 2225822264.
D.R. Abernethy, N.M. Soldatov, Structure-functional diversity of
human L-type Ca2+ channel: perspectives for new pharmacological targets, J. Pharmacol. Exp. Ther. 300 (2002) 724728.
A.H. Kim, C.T. Sheline, M. Tian, T. Higashi, R.J. McMahon,
R.J. Cousins, D.W. Choi, L-type Ca(2+) channel-mediated
Zn(2+) toxicity and modulation by ZnT-1 in PC-12 cells (1),
Brain Res. 886 (2000) 99107.
S.L. Sensi, L.M. Canzoniero, S.P. Yu, H.S. Ying, J.Y. Koh,
G.A. Kerchner, D.W. Choi, Measurement of intracellular free
zinc in living cortical neurons: routes of entry, J. Neurosci. 17
(1997) 95549564.
C. Nolte, A. Gore, I. Sekler, W. Kresse, M. Hershnkel, A.
Homann, H. Kettenmann, A. Moran, ZnT-1 expression in
astroglial cells protects against zinc toxicity and slows the
accumulation of intracellular zinc, Glia (2004) in press.
L.A. Gaither, D.J. Eide, Eukaryotic zinc transporters and their
regulation, Biometals 14 (2001) 251270.
R.J. Cousins, R.K. Blanchard, J.B. Moore, L. Cui, C.L. Green,
J.P. Liuzzi, J. Cao, J.A. Bobo, E.D. Harris, Regulation of zinc
metabolism and genomic outcomes cellular transporters for zinc,
J. Nutr. 133 (2003) 1521S1526S.
S. Devergnas, F. Chimienti, N. Naud, A. Pennequin, Y. Coquerel,
J. Chantegrel, A. Favier, M. Seve, Dierential regulation of zinc
eux transporters ZnT-1, ZnT-5 and ZnT-7 gene expression by
zinc levels: a real-time RT-PCR study, Biochem. Pharmacol. 68
(2004) 699709.
R.D. Palmiter, S.D. Findley, Cloning and functional characterization of a mammalian zinc transporter that confers resistance to
zinc, EMBO J. 14 (1995) 639649.
S.J. Langmade, R. Ravindra, P.J. Daniels, G.K. Andrews, The
transcription factor MTF-1 mediates metal regulation of the
mouse ZnT1 gene, J. Biol. Chem. 275 (2000) 3480334809.
Y.B. Nitzan, I. Sekler, M. Hershnkel, A. Moran, W.F. Silverman, Postnatal regulation of ZnT-1 expression in the mouse brain,
Brain Res. Dev. Brain Res. 137 (2002) 149157.
J.P. Dehaye, Regulation by purinergic agonists of zinc uptake by
rat submandibular glands, J. Trace Elem. Med. Biol. 9 (1995) 94
101.
P. Arslan, F. Di Virgilio, M. Beltrame, R.Y. Tsien, T. Pozzan,
Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas.
A new, membrane-permeant chelator of heavy metals reveals that
these ascites tumor cell lines have normal cytosolic free Ca2+, J.
Biol. Chem. 260 (1985) 27192727.
D.M. Fine, C.F. Lo, L. Aguillar, D.L. Blackmon, M.H. Montrose, Cellular chloride depletion inhibits cAMP-activated electro-

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]
[36]
[37]

[38]

[39]

genic chloride uxes in HT29-18-C1 cells, J. Membr. Biol. 145

(1995) 129141.
I. Sekler, R.S. Lo, R.R. Kopito, A conserved glutamate is
responsible for ion selectivity and pH dependence of the
mammalian anion exchangers AE1 and AE2, J. Biol. Chem. 270
(1995) 2875128758.
A. Moran, R.J. Turner, Secretagogue-induced RVD in HSY cells
is due to K+ channels activated by Ca2+ and protein kinaseC, Am.
J. Physiol. 265 (1993) C1405C1411.
F.L. Graham, A.J. van der Eb, A new technique for the assay of
infectivity of human adenovirus 5 DNA, Virology 52 (1973) 456
467.
M. Hershnkel, A. Moran, N. Grossman, I. Sekler, A zincsensing receptor triggers the release of intracellular Ca2+ and
regulates ion transport, Proc. Natl. Acad. Sci. USA 98 (2001)
1174911754.
G. Grynkiewicz, M. Poinie, R.Y. Tsien, A new generation of
Ca++ indicators with greatly improved uorescence properties, J.
Biol. Chem. 260 (1985) 34403450.
M.A. Markwell, S.M. Haas, L.L. Bieber, N.E. Tolbert, A
modication of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples, Anal. Biochem. 87
(1978) 206210.
I. Sekler, A. Moran, M. Hershnkel, A. Dori, A. Margulis, N.
Birenzweig, Y. Nitzan, W.F. Silverman, Distribution of the zinc
transporter ZnT-1 in comparison with chelatable zinc in the
mouse brain, J. Comp. Neurol. 447 (2002) 201209.
R.D. Palmiter, Protection against zinc toxicity by metallothionein
and zinc transporter1, Proc. Natl. Acad. Sci. USA 101 (2004)
49184923, Epub. 2004 Mar. 4923.
E. Ohana, D. Segal, R. Palty, D. Ton-That, A. Moran, S.L.
Sensi, J.H. Weiss, M. Hershnkel, I. Sekler, A sodium zinc
exchange mechanism is mediating extrusion of zinc in mammalian
cells, J. Biol. Chem. 279 (2004) 42784284.
R. Cohen, L.A. Elferink, D. Atlas, The C2A domain of
synaptotagmin alters the kinetics of voltage-gated Ca2+ channels
Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type), J. Biol. Chem. 278
(2003) 92589266.
R.J. Cousins, R.J. McMahon, Integrative aspects of zinc transporters, J. Nutr. 130 (2000) 1384S1387S.
G. Rudnick, ATP-driven H+ pumping into intracellular organelles, Annu. Rev. Physiol. 48 (1986) 403413.
T.J. Kamp, J.W. Hell, Regulation of cardiac L-type calcium
channels by protein kinase A and protein kinase C, Circ. Res. 87
(2000) 10951102.
E. Pravettoni, A. Bacci, S. Coco, P. Forbicini, M. Matteoli, C.
Verderio, Dierent localizations and functions of L-type and Ntype calcium channels during development of hippocampal
neurons, Dev. Biol. 227 (2000) 581594.
R.J. McMahon, R.J. Cousins, Regulation of the zinc transporter
ZnT-1 by dietary zinc, Proc. Natl. Acad. Sci. USA 95 (1998) 4841
4846.