(Received for publication, September 30, 1994; and in revised form, November 21, 1994)
From the
Electrical activity initiates a program of selective gene
expression in excitable cells. Although such transcriptional activation
is commonly attributed to depolarization-induced changes in
intracellular Ca, zinc represents a viable
alternative given its prominent role as a cofactor in DNA-binding
proteins coupled with evidence that Zn
can enter
excitable cells in a voltage-dependent manner. Here it is shown that
Zn
entry into heart cells depends upon electrical
stimulation and occurs via dihydropyridine-sensitive Ca
channels. The addition of extracellular Zn
to
spontaneously depolarizing GH3 pituitary tumor cells induced the
expression of a reporter gene driven by the metallothionein promoter,
an effect that was prevented by exposure to dihydropyridine
Ca
channel blockers. Thus, Zn
influx through L-type Ca
channels can mediate
voltage-dependent gene expression.
Zinc plays prominent catalytic and structural roles in various
key proteins, including RNA polymerase and zinc-finger transcription
factors(1, 2) . In addition, zinc may also function
dynamically as a biological second messenger(3) . Exposure to
extracellular zinc can quickly and selectively turn on the expression
of metallothionein genes in intact cells(4, 5) ,
suggesting that changes in intracellular Zn concentration can influence gene expression. Remarkably little is
known about intracellular Zn
or the mechanisms that
control its concentration, although several lines of evidence suggest
that Zn
enters neurons and cardiac myocytes through
specific voltage-activated conductance pathways (6, 7, 8) . To clarify the nature of these
pathways, we measured intracellular free Zn
concentration and membrane currents in heart cells. The results
indicated that zinc is indeed tightly regulated and that the metal
enters excitable cells through dihydropyridine-sensitive calcium
channels. To investigate whether the zinc that enters through calcium
channels acts as a second messenger in transcriptional activation, we
measured the activity of a reporter gene linked to the metallothionein
promoter. Exposure to zinc induced expression of the gene in GH3 cells,
but not when calcium channels were blocked pharmacologically. The
effects of electrical activity on transcription enable the genetic
program of a cell to be shaped by its history of
excitation(9, 10, 11, 12, 13) .
The present results indicate that Zn
can mediate
excitation-transcription coupling, and thus this ion merits
consideration as a possible mediator of processes as diverse as memory
and heart failure(14, 15, 16) .
where X refers to Ca or
Zn
, K
` is the
apparent dissociation constant for X, R
is the maximum fluorescence ratio
at saturating [X], and R
is
the fluorescence ratio in the absence of X. Furthermore, K
` = K
where
is
the ratio of the 510 nm fluorescence measured with 380 nm excitation in
the absence of X to that recorded at saturating levels of X and K
is the actual dissociation
constant for X binding to fura-2(19, 20) .
From the in vitro calibration experiments shown in Fig. 1A, K
` was
determined to be 6.4 nM, with R
= 0.56 and R
= 2.8. The
corresponding values for Ca
were as follows: K
` = 3.5 µM, R
= 0.53, and R
= 7.0. After corrections for the
values, the true K
values equaled 3.0 nM (Zn
) and 310 nM (Ca
).
Figure 1:
Calibrations. A, in vitro calibration of fura-2 with Ca in the absence of
Zn
(
) and Zn
in the absence
of Ca
(
). The measured ratios of the
fura-2-dependent fluorescence are plotted against the estimated
[Ca
] and [Zn
]. B, in vitro calibration with varied
[Zn
] in the presence of 1 mM total
CaCl
added to the calibrating solutions (yielding
25-100 nM free [Ca
]). The
measured ratio of the fura-2-dependent fluorescence is plotted for
various [Zn
]. The line corresponds
to the predicted ratio using a competitive binding equation (see ).
Competition between Ca and Zn
binding to fura-2 was explored by comparing the measured
fluorescence ratio with that predicted by a competitive binding
equation when the [Zn
] was varied in the
presence of 1 mM CaCl
(i.e. the solutions
contained (mM): 100 KCl, 10 HEPES, 10 EGTA, 20 NTA, 1
CaCl
, 10
fura-2 and varied amounts of
ZnCl
(pH 7.15)). The competitive binding equation used
was:
where the various constants used were estimated from the
separate calibrations with either Ca or
Zn
alone. As seen from inspection of Fig. 1B, excellent agreement between the measured and
predicted ratio (R) was observed, indicating that the
competitive binding equation accurately predicts the fluorescence ratio
in conditions where both Ca
and Zn
are present.
Figure 2:
Stimulation dependence of
Zn influx. A, representative records of
fluorescence and force from a rat cardiac trabecula loaded with fura-2.
The left-most records illustrate fluorescence changes at 340
nm and 380 nm (Ca
transients) and the associated
contraction with 0.5 mM Ca
in the perfusate
during stimulation at 0.5 Hz. The changes in fluorescence during
quiescence and stimulation in the presence of 0.1 mM Ca
± 20 µM Zn
are shown in subsequent records. B, changes in the 340
nm/380 nm fluorescence ratio (left axis) and
[Zn
] (right axis) during
quiescence and electrical stimulation in the same experiment as in A. Data points were recorded every 30 s. C,
representative records from a control experiment subjected to the same
protocol as the preparation in panel A, but without exposure
to Zn
. D, changes in the 340/380 ratio for
the experiment shown in C.
Figure 7:
Transcriptional activation by
Zn influx. A, reporter gene activity in GH3
cells transfected with pMT-luc and treated with or without 20
µM ZnCl
± 20 µM nitrendipine (NT). Luciferase activity measured as
relative light units was normalized by protein content in each plate.
Results are expressed as percentage of the normalized luciferase
activity in the 0 Zn
group. The 20 µM Zn
group was statistically different from all
the other groups (n = 8, p < 0.05). B, reporter gene activity in GH3 cells transfected identically
as in A. The two groups (each n = 6) were
incubated with either 20 µM ZnCl
and 20
µM nitrendipine, or with 20 µM ZnCl
, 20 µM nitrendipine, and 1
µM dexamethasone (DEX). Results are expressed as
luciferase activity measured in relative light units normalized by
protein content. Mock-transfected cells (rightcolumn, n = 6) exhibited very low
luminescence levels. C, changes of fluorescence ratios in GH3
cells loaded with the AM-ester form of fura-2(22) . Each data
point represents the mean ± S.D. of four
experiments.
Although most commonly used to measure
[Ca], fura-2 actually binds Zn
much more avidly than Ca
but, unlike other
heavy metals, Zn
does not quench the
dye(27) . Fig. 1A shows in vitro calibration curves for pure Zn
(opencircles) and pure Ca
solutions (filledcircles). Fura-2 binds Zn
with a 100-fold higher affinity than it binds
Ca
, although the maximal fluorescence ratio produced
by the reaction is lower. Fura-2 is a useful Zn
indicator even under mixed ionic conditions designed to mimic
those in the cytosol; in the presence of background free
[Ca
] of 25-100 nM (calculated from the buffering equations above given the addition
of 1 mM total calcium to the calibrating solutions), fura-2
remains sensitive to changes of [Zn
] in the
subnanomolar range (Fig. 1B).
Rat ventricular
trabeculae were loaded with fura-2 to measure the intracellular
concentration of Zn in cardiac myocytes (19, 21) . Electrical stimulation in physiological
solution containing 0.5 mM Ca
elicited
typical Ca
-dependent transients of fura-2
fluorescence and contractile force (19) (Fig. 2A, left-most
records). Calcium transients and force were eliminated by
decreasing extracellular Ca
to 0.1
mM(20) . The subsequent addition of 20 µM Zn
(which approximates the total concentration
of zinc in mammalian blood; (1) and (2) ) to the
external solution produced little change in fluorescence in the absence
of electrical stimulation (5-35 min). Nevertheless, the
fluorescence at 340 nm (F
) increased rapidly and
progressively when 0.5 Hz electrical stimulation was initiated; the
fluorescence at 380 nm (F
) changed in parallel
but in the opposite direction, consistent with an increase in
[Zn
]
(Fig. 1). The
absence of any rise in force verifies that the changes in fura-2
fluorescence are due to Zn
influx rather than to an
increase in basal [Ca
]
; unlike
Ca
, Zn
binds to troponin very
weakly and does not activate contraction(28) . Fig. 2B shows the time course of the changes in the
Zn
-dependent fluorescence ratio (left axis)
or [Zn
]
(right axis)
in this muscle before, during, and after a 30-min period of electrical
stimulation. Intracellular Zn
remains very low
(<0.1 nM) during quiescence, but increases by more than 1
order of magnitude within 10-15 min of the onset of stimulation.
The fluorescence ratio declined slowly after the cessation of
stimulation, suggesting a gradual extrusion of the accumulated
intracellular Zn
by unknown mechanisms.
Several
lines of evidence support our interpretation that the changes in fura-2
fluorescence in Fig. 2(A and B) arise
predominantly from an increase in
[Zn]
. First, the
stimulation-dependent changes of fluorescence in the absence of
Zn
were much smaller (Fig. 2, C and D), corresponding to a modest rise in
[Ca
]
(from 11 to 33
nM, n = 3). Second, the sharp
stimulation-dependent increase in the fluorescence ratio could be
truncated dramatically by exposure to TPEN, a specific cell-permeant
chelator of heavy metals (Fig. 3)(24) . Results similar
to those in Fig. 3were obtained in two other experiments using
TPEN in the presence of Zn
; TPEN had no effect when
applied in the absence of Zn
. Finally, the initial
rate of rise of the fluorescence ratios upon stimulation increased as
the extracellular Zn
concentration was raised over
the range from 0.5 to 20 µM (Fig. 4). The
observation of appropriate changes in fluorescence even at
submicromolar extracellular Zn
concentrations is
significant, because most of the 15-20 µM zinc in
blood is likely to be bound to proteins(1, 2) .
Figure 3:
Representative experiment showing the
effect of the Zn-chelating agent TPEN (30
µM) on the changes of fluorescence ratios in a
fura-2-loaded trabecula.
Figure 4:
Dynamics of intracellular Zn uptake as a function of extracellular
[Zn
]. The changes in the 340/380
fluorescence ratios, measured over the first 3 min of electrical
stimulation, are plotted. n = 3 in the first three data
points ([Zn
]
=
0.5, 2, and 10 µM) and n = 6 at
[Zn
]
= 20
µM.
The
striking stimulation-dependence of Zn influx was a
consistent finding. Fig. 5shows pooled data from four
experiments in which Zn
was added in the absence of
any drug (open circles).
[Zn
]
did not change
significantly until electrical stimulation was initiated at 35 min, but
then it increased rapidly from <0.4 nM to >2.0
nM. The brisk response to stimulation suggests activation of a
voltage-dependent pathway for Zn
influx. One
candidate for such a pathway is the L-type Ca
current. Although transition metals are generally viewed as
Ca
channel blockers(29, 30) , action
potential experiments and single-channel recordings (6, 7, 8) suggest that Zn
may actually be capable of permeating L-type Ca
channels. The idea that Zn
enters heart cells
via L-type Ca
channels was tested by examining the
effect of a dihydropyridine Ca
channel blocker on the
response to electrical stimulation (Fig. 5, filled
circles). Nitrendipine (10 µM) markedly attenuated
the influx of Zn
during electrical stimulation. The
residual change in fluorescence closely resembles that seen in the
absence of Zn
(e.g.Fig. 2D).
Figure 5:
Effects of nitrendipine on
Zn influx. A, pooled data showing
340 nm/380 nm fluorescence ratios (left y axis) and
corresponding Zn
concentrations (righty axis) of drug-free experiments (mean ± S.E., n = 4,
) as well as experiments during which 10
µM nitrendipine was added (at 20 min, n =
4,
) to the perfusate that contained 0.1 mM Ca
at all times. The inhibition of
Zn
influx by nitrendipine, as expressed by the
blunted increase of fluorescence ratio, becomes highly significant
during electrical stimulation (p < 0.0001 by analysis of
variance; interval 35-65 min).
To confirm the idea that
Zn can enter through voltage-dependent Ca
channels, we measured membrane currents through L-type channels
in single rat ventricular myocytes (22, 23) . Fig. 6shows currents from one such experiment in which the
charge carrier was changed from 5 mM Ca
(panel A) to 20 mM Zn
(panel B). Millimolar concentrations of divalent cations
are needed to resolve inward membrane currents, given that L-type
Ca
channels rely on multi-ion occupancy to boost
conductance while maintaining selectivity(29) . Ca
and Zn
both supported inward currents, which
were sensitive to the dihydropyridine Ca
channel
blocker PN-200-110. The Zn
currents are surprisingly
robust, demonstrating directly that Zn
can permeate
L-type Ca
channels. Little or no net inward current
was elicited when both Ca
and Zn
were omitted from the bathing solution (panel C).
Figure 6:
Dihydropyridine-sensitive
Ca, Zn
, and zero-divalent currents
recorded in a rat ventricular myocyte. A, PN-200-110-sensitive
current (i.e. the current recorded in the presence of 20
µM PN-200-110 subtracted from the current in the absence
of the Ca
channel blocker) in 5 mM external
Ca
during a voltage step from -35 to 0 mV. B, the PN-200-110-sensitive current recorded with 20 mM external Zn
. C, the
PN-200-110-sensitive current recorded in the absence of added
Zn
or Ca
. The solid lines represent the zero current level.
Zinc is an obligatory cofactor for various transcriptional activator
DNA-binding proteins as well as many key enzymes, and several lines of
evidence indicate that changes in intracellular Zn may suffice to trigger selective gene expression(3) . In
particular, the metallothionein gene responds to heavy metals such as
Zn
with a rapid transcriptional
activation(4, 5) . Given our observations that
Zn
can permeate Ca
channels and
thereby enter excitable cells in a voltage-dependent manner, we
determined whether this pathway could mediate excitation-transcription
coupling. A genetic construct (pMT-luc) consisting of the mouse
metallothionein I gene promoter (4, 5) fused to the
firefly luciferase gene (31) was transfected into GH3 pituitary
tumor cells, which are known to undergo spontaneous and repetitive
Ca
channel-dependent action potentials in
culture(32, 33) . We chose GH3 cells for these studies
because the rationale of the experiments should be generalizable to all
cells that have active L-type Ca
channels, and
because no satisfactory heart cell line is available. After
transfection, cells were randomly divided into four groups with or
without 20 µM Zn
and/or 20 µM nitrendipine added to medium that contained physiological
concentrations of CaCl
. Fig. 7A shows the
relative levels of expression of the luciferase reporter gene in the
four groups. The simple addition of 20 µM Zn
sufficed to turn on the metallothionein promoter; the 2-fold
enhancement of reporter gene expression agrees with previous data at
this low [Zn
] (34) . The
Zn
-induced transactivation was prevented by
concomitant incubation of the cells with nitrendipine. Cells in which
Zn
-dependent metallothionein gene induction had been
prevented by nitrendipine were still capable of responding to
dexamethasone (Fig. 7B), a steroid hormone known to
activate the metallothionein promoter by metal-independent
routes(35) . Fig. 7C shows additional evidence
that the effect of nitrendipine reflects inhibition of Zn
influx rather than a toxic effect: fura-2 fluorescence in GH3
cells increases when they are exposed to Zn
(filled symbols), an effect that is blocked by
nitrendipine (open symbols).
Excitation-transcription
coupling represents an elementary pathway whereby the electrical
activity of a cell feeds back upon and shapes the genetic program of
that cell. When viewed in this manner, excitation-transcription
coupling underlies several crucial cellular processes. Here it has been
demonstrated that the influx of Zn via
voltage-dependent Ca
channels can turn on gene
expression in a simple model system. While these results illustrate one
potential consequence of voltage-dependent Zn
influx,
they leave unresolved the question of the relative importance of this
ion in the various physiological systems where electrical activity is
known to alter gene expression. Further studies will be required to
elucidate the role of voltage-dependent Zn
influx in vivo in processes as varied as differentiation and
memory(14, 15, 16) . Given the diversity of
Zn
-dependent enzymes, which control many processes
other than transcription, there is good reason to expect a variety of
biological effects of electrically stimulated Zn
uptake.