©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Excitation-Transcription Coupling Mediated by Zinc Influx through Voltage-dependent Calcium Channels (*)

(Received for publication, September 30, 1994; and in revised form, November 21, 1994)

Dan Atar (§) Peter H. Backx Melissa M. Appel Wei Dong Gao Eduardo Marban (¶)

From the Department of Medicine, Division of Cardiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) .


MATERIALS AND METHODS

Fura-2 Calibration for the Estimation of [Ca] and [Zn]

Fura-2 calibrations with Ca were performed at 22 °C using different concentrations of Ca-buffered solutions with EGTA containing (mM): 120 KCl, 10 HEPES, 10 EGTA, 10 fura-2, and varied quantities of CaCl(2) (pH 7.15). Similarly, calibration of fura-2 fluorescence with Zn was achieved using varied concentrations of Zn-buffered solutions with NTA (^1)and EGTA containing (mM): 100 KCl, 10 HEPES, 10 EGTA, 20 NTA, 10 fura-2, and varied amounts of ZnCl(2) (pH 7.15). The [Ca] and [Zn] were estimated by iteratively solving the non-linear set of equilibrium binding equations describing Ca and Zn binding to the added ligands (17) using published dissociation constants(18) . The ratio (R) of the background-subtracted fluorescence at 510 nm recorded with excitation at 340 nm to that recorded at 380 nm excitation at different concentrations of X were fit to ,

where X refers to Ca or Zn, K` is the apparent dissociation constant for X, R(X) is the maximum fluorescence ratio at saturating [X], and R(min) is the fluorescence ratio in the absence of X. Furthermore, K` = Kbeta(X) where beta(X) 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(D)` was determined to be 6.4 nM, with R = 0.56 and R = 2.8. The corresponding values for Ca were as follows: K(D)` = 3.5 µM, R = 0.53, and R = 7.0. After corrections for the beta values, the true K(D) 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 (bullet) and Zn in the absence of Ca (circle). 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(2) 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(2) (i.e. the solutions contained (mM): 100 KCl, 10 HEPES, 10 EGTA, 20 NTA, 1 CaCl(2), 10 fura-2 and varied amounts of ZnCl(2) (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.

Fluorescence and Force Measurements in Cardiac Muscle

Thin ventricular trabeculae (1.8 mm times 150 µm times 50 µm) were dissected from the right ventricles of 2-3-month-old male rats (LBN-F1 strain, Harlan, Indianapolis, IN). The muscles were mounted on the stage of an inverted microscope between a force transducer and a micromanipulator and continuously superfused with a solution containing (in mM) 120 NaCl, 4.5 KCl, 1.2 MgCl(2), 1 NaH(2)PO(4), 20 NaHCO(3), 0.1 CaCl(2), and 10 glucose, equilibrated with 95% O(2) and 5% CO(2) to a pH of 7.4 (22-23 °C). Electrical field stimulation at 0.5 Hz was achieved across two platinum electrodes within the perfusion bath. Fura-2 potassium salt (Molecular Probes, Eugene, OR) was loaded into the muscle by microinjection, as described previously(19, 21) . Excitation ultra-violet (UV) light from a mercury lamp was bandpass-filtered at 340, 357, or 380 nm and projected onto the muscle via a 10times objective. Emitted fluorescent light was transmitted to a photomultiplier, filtered at 100 Hz (3 dB), and stored digitally for later analysis. [Zn](i) was estimated using the competitive binding equation. The algorithm for estimating [Zn](i) involved three steps. 1) Prior to the addition of Zn at low external [Ca] (0.1 mM), the fluorescence ratio was recorded. The [Ca](i) was estimated from this ratio by assuming that the [Zn] was zero. 2) The [Ca](i) recorded prior to the application of Zn was assumed to remain fixed throughout the experiment. 3) Following the application of external Zn, we observed the fluorescence ratio and estimated [Zn](i) using the competitive binding equation above (). The assumption in step 2 may not be entirely valid, since small increases in basal Ca-dependent fluorescence are observed in the absence of external Zn (e.g.Fig. 2D). Such Ca-dependent changes would be minimized by the concomitant presence of Zn, for two reasons. First, Zn competes effectively with Ca for binding to fura-2 (Fig. 1); second, the presence of extracellular Zn will antagonize Ca entry into the cells(6, 7, 8) . To the extent that [Ca](i) in fact rises in the presence of Zn, the calibrated values of [Zn](i) represent an upper-limit estimate.



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.



Membrane Current Recordings

Enzymatically isolated rat ventricular myocytes were placed into a perfusion bath located on the stage of an inverted microscope and superfused with solution containing (in mM): 140 NaCl, 5 KCl, 10 HEPES (pH 7.4), 1 MgCl(2), 5 CaCl(2), or 20 ZnCl(2), and 10 dextrose. At the end of the experiment, the Ca channel blocker PN-200-110 (20 µM) was added to block current through L-type Ca channels and to enable calculation of the dihydropyridine-sensitive current. Membrane currents were recorded using the whole-cell patch clamp configuration(22) . The intracellular solution contained (in mM): 120 KCl, 5 NaCl, 1 MgCl(2), 10 HEPES (pH 7.1 with KOH), 2.5 MgATP, and 2 EGTA. Further details of the experimental protocol have been described previously(23) .

Reporter Gene Experiments

The genetic construct pMT-luc was created by removing a 1.1-kilobase fragment from pBPV which contains the mouse metallothionein promoter (4, 5) and inserting it into pGL2-Basic upstream of the firefly luciferase gene(31) . Twenty-four hours before transfection, 2-3 times 10^6 cells were plated on 35-mm Petri dishes. Transfections (n = 8) were done in duplicate or triplicate on three different days. GH3 cells were transfected using a liposome-mediated method with pMT-luc (1 µg/plate). Cells were transfected before being randomly divided into treatment groups to avoid possible errors due to different transfection efficiencies. After 5 h of transfection, the cells were incubated in Opti-MEM I reduced serum medium (Life Technologies, Inc.) overnight with or without 20 µM Zn or 20 µM nitrendipine. Eighteen hours later the cells were lysed with Reporter lysis buffer and assayed for luciferase activity on a luminometer (25) as well as for protein content, using the Micro BCA protein assay method (Pierce). Background luciferase activity as assessed in mock transfection was subtracted from all luciferase activity measurements, normalized for protein content, and expressed as mean ± S.E. An unpaired Student's t test was performed to compare means of the groups. For Fig. 7B, 2-3 times 10^6 cells were plated on 35-mm Petri dishes 24 h before transfection (n = 6). Incubation with 20 µM Zn and 20 µM nitrendipine was performed as outlined under ``Materials and Methods'' (membrane current recordings) in half of the plates, whereas the other half was incubated with 20 µM Zn, 20 µM nitrendipine, and 1 µM dexamethasone. Luciferase activity was measured and analyzed as described in (25) , except that the results are expressed as relative light units/µg protein (y axis in Fig. 7B).



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(2) ± 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(2) and 20 µM nitrendipine, or with 20 µM ZnCl(2), 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.



Fluorescence Measurements in GH3 Cells

GH3 cells were plated on 35-mm dishes (3 times 10^6 cells/dish) 24 h before the experiment. Autofluorescence was measured after washing the cells in the same physiological solution as used for trabeculae experiments (19, 21) . Cells were then exposed to fura-2-AM (5 µM in the loading buffer mixed with 1 µM pluronic acid) for 1 h at 22 °C. Fluorescence was measured as described(19, 21) , with the Petri dishes occupying the space of the perfusion bath on the stage of the microscope. Because the fura-2-AM may not have been completely hydrolyzed(26) , the fluorescence ratios in Fig. 7C have not been converted to [Zn](i). To assess the significance of the differences in fluorescence ratios, a Student's t test was utilized.


RESULTS AND DISCUSSION

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](i) (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](i); 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](i) (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](i). 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](i) (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](i) 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, circle) as well as experiments during which 10 µM nitrendipine was added (at 20 min, n = 4, bullet) 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 Caand 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(2). 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.


FOOTNOTES

*
This work was supported by grants from the Swiss National Science Foundation (to D. A.) and the American Heart Association (to W. D. G.) and National Institutes of Health Grant R01 HL 44065 (to E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Division of Cardiology, Dept. of Medicine, Basel University Hospital, CH-4031 Basel, Switzerland.

To whom correspondence should be addressed: Dept. of Medicine, Division of Cardiology, The Johns Hopkins University School of Medicine, 844 Ross Bldg., 720 N. Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-2776; Fax: 410-955-7953; marban{at}welchlink.welch.jhu.edu.

(^1)
The abbreviations used are: NTA, nitrilotriacetic acid; TPEN, N, N, N`,N`-tetrakis-(2-pyridylmethyl)ethylenediamine.


ACKNOWLEDGEMENTS

We thank Dr. Michelle Azan-Backx and Maria Janecki for assistance with some of the experiments and Drs. Jeremy Berg, Chi V. Dang, Timothy J. Kamp, and Gordon Tomaselli for comments on the manuscript.


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