Neuroscience Letters, 117 (1990) 117-122 Elsevier Scientific Publishers Ireland Ltd.
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NSL 07126
Zn 2 + blocks the voltage activated calcium current o f Aplysia neurons D . B f i s s e l b e r g 1,2 M . L . E v a n s 1, H . R a h m a n n 2 a n d D . O . C a r p e n t e r 1 1Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, N Y 12209-0509 (U.S.A.), School of Public Health, State University of New York at Albany, Albany, N Y 12237 (U.S.A.) and 2Universitiit Stuttgart-Hohenheim, Institut fiir Zoologic, Stuttgart (F.R.G.)
(Received 4 January 1990; Revised version received 2 April 1990; Accepted 21 May 1990) Key words." Zinc; Calcium current; Voltage clamp; Fixed charge site
We have investigated the effect of Zn2+ on voltage-activated calcium currents of Aplysia neurons, using conventional two-electrode voltage-clamp techniques. The peak of these currents was reversibly reduced by Zn 2+ (50% reduction at 3.75 mM; total block at 20 mM), while the current-voltage relation and the activation and inactivation curves were shifted to depolarized voltages. The effects of Zn 2+ were concentration-dependent. The Hill coefficient was 1.62. The high concentrations required, the shift of the currentvoltage relation and the effects on activation and inactivation are best explained by a charge-screening effect combined with a specific binding site for Zn 2÷ near the entrance of the channel.
Zinc ions have a variety o f actions on nerve cells. Y o k o y a m a et al. [20] r e p o r t e d t h a t even small a m o u n t s o f this heavy m e t a l c a t i o n are extremely toxic to neurons. O n the o t h e r h a n d there are n a t u r a l l y high c o n c e n t r a t i o n s o f zinc a t t a c h e d to m e m branes, especially in the n e r v o u s system [2], while at s o m e sites including h i p p o c a m pus a n d p i r i f o r m cortex there are high c o n c e n t r a t i o n s o f Z n 2+ in p r e s y n a p t i c terminals, t o g e t h e r with evidence t h a t Z n 2+ is released a l o n g with t r a n s m i t t e r substances [1, 18]. Z n 2+ has been s h o w n to m o d u l a t e t r a n s m i t t e r release [15, 19] a n d b l o c k channels a c t i v a t e d by N - m e t h y l - D - a s p a r t a t e [1 1, 16]. In a d d i t i o n Gilly a n d A r m s t r o n g [7, 8] r e p o r t e d t h a t Z n 2+ alters the kinetics o f s o d i u m a n d p o t a s s i u m currents. N u m e r o u s studies have s h o w n t h a t d i v a l e n t c a t i o n s such as C o 2+ a n d C d 2+ are p o t e n t a n t a g o n i s t s o f v o l t a g e - d e p e n d e n t calcium currents on a variety o f nerve cells [see 9]. T h e r e has, however, been little s t u d y o f the effect o f Z n 2+ despite the fact that, unlike several o t h e r divalents, Z n 2 + has a p h y s i o l o g i c function in the n e r v o u s system [1, 19]. A b d o m i n a l g a n g l i a o f A p l y s i a californica (120--200 g) were r e m o v e d a n d p i n n e d to a S y l g a r d - c o a t e d c h a m b e r in artificial s e a w a t e r ( A S W ) . The capsule was then Correspondence.. D. Biisselberg. Present address: Johannes-Gutenberg Universit/it Mainz, II. Institut f/Jr Physiologie und Pathophysiologie, Abteilung Biophysik, Duesbergweg 6, D-6500 Mainz, F.R.G.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
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opened, the connective tissue sheath removed and the neurons exposed. The cells were washed with ASW for at least 30 rain before penetration. All solutions were applied via a rapid perfusion system that results in complete change of the solution surrounding the cell under investigation within 1 s [I 7]. Neurons were impaled with two independent microetectrodes (4-8 Mr2). In order to record voltage-activated calcium currents the ASW was replaced with a solution of the following composition (in mM): MgC12 45, KCI 10, CaC12 20, tetraethylammonium bromide 200 [21], 4-aminopyridine 1 [10], Tris-(hydroxymethyl)amino-methane-hydrochloride (Tris) 234, and Tris base 59 raM. Final pH was adjusted to 7.6. The voltage electrode was filled with 3 M potassium acetate and the current electrode was filled with 4 M cesium chloride (Aldrich, 99.9995% purity) to block the outward potassium currents. Zinc (II) chloride (Aldrich, 99.999% purity) was dissolved directly in the seawater replacement solution just before each experiment to avoid precipitation of zinc salts. No precipitation was observed with Zn 2 + concentrations of 40 mM or less. Neurons were routinely clamped at - 4 0 mV, which is close to the resting membrane potential. The standard voltage step was to + 20 mV for 70 ms. All currents were recorded on tape via a digitizing unit and analyzed using a P/3 protocol [5] for leakage subtraction. This procedure avoids errors due to activation of chloride currents by the hyperpolarization [6]. B
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Voltage jumps were not applied at intervals more frequent than 20 s, and under these circumstances the induced currents were of constant amplitude. The peak amplitude of the voltage-dependent calcium current was reduced in a dose-dependent fashion when the preparation was perfused with ASW to which zinc chloride (0.2520 mM) was added. Fig. 1A shows currents recorded in different concentrations of Zn 2+. Note that the time to peak current became greater with increasing Zn 2+ concentrations. Without Zn 2+ the current peaked at 3-5 ms, but in 20 mM Zn 2+ the peak occurred after more than 20 ms. Fig. 1B shows the dose-response relation obtained from 65 applications of Zn 2+ to more than 30 different neurons. The threshold concentration for effects of Zn 2+ was about 0.25 mM, while 5 m M gave about 58% reduction of peak current. Near complete ( > 80%) inhibition was observed at Zn 2+ concentrations greater than 10 mM. The calculated Hill coefficient was 1.62. These studies were done on a variety of identified (RB, R2, R15, L2-6) and unidentified neurons of the abdominal ganglion, and no differences were found in either the characteristics of the voltage-dependent calcium current or the effects of Zn 2+ among these cells. Fig. 2 shows the time course of Zn 2+ blockade and reversal upon perfusion of control ASW. The calcium current reduction was already apparent with the first voltage jump applied 20 s after initiation of the perfusion of Zn 2+. It increased to reach a steady-state within 5-10 min, and recovered to within 10% of control in all but 2 of 65 experiments with an approximately similar time course. The time required to reach steady state and recovery (especially recovery) was greater with higher concentrations of Zn 2+. With 20-40 m M Zn 2+ complete recovery sometimes required 30-45 min. The effects of Zn 2+ on calcium currents might be direct channel blockade or rather APPLICATION 100. n2+5mM 80,
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a consequence of an alteration of the kinetics of the response. Fig. 3A shows the effects of three concentrations of Zn 2+ (1, 2.5 and 5 m M for 10 rain) on the currentvoltage relation. In the control the largest current was elicited by a j u m p to + 20 mV. With jumps to less positive potentials the current amplitude was smaller, reflecting the voltage-dependent activation of the calcium current, while with jumps to more positive values the current was reduced as a result of approaching the equilibrium potential for calcium. In the presence of Zn 2 + the voltage at which the maximal current was generated shifted in a depolarizing direction in a dose-dependent fashion. In 5 m M Zn 2+ the peak current was elicited at a potential about 15 mV more depolarized than control. Fig. 3B illustrates the voltage dependence of activation and inactivation in a con-
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121
trol ASW containing half the normal Ca 2+ concentration and in the presence of 5 m M Zn 2+ and half normal Ca 2+ ASW. The lower Ca 2+ concentration was used in this experiment to maximize the effect of Zn 2+ on the kinetics of the response. Both activation and inactivation curves were shifted in depolarizing directions in the presence of Zn 2+. The concentration of Zn 2+ required for effect, the rapid onset and washout, and the alteration of the current-voltage relation and of activation and inactivation strongly suggest a binding site external to the calcium channel. There are two possible mechanisms by which this might occur: a general binding of cations to negative charges of the surface (charge screening) or a specific binding to (a) site(s) external to the channel. General charge screening should be not specific for a single channel type, while a more specific site external to a channel might be more selective. The high concentration of Zn 2+ used in this study, which probably does not occur physiologically, makes a general charge screening effect most likely. The increase of time to the peak of the current, the shift of the current-voltage relation and of activation and inactivation to depolarized voltages would all be expected as a result of charge screening. This has been demonstrated to occur when Ca 2+ concentration is increased [4, 12, 13]. However, when Ca 2+ concentration is increased the current flowing through Ca 2+ channels is increased, in addition to the changes induced in the kinetics of the response. With Zn 2+ the kinetics change, but the current decreases. It is, however, difficult to explain the degree of channel blockade solely on the basis of charge screening, which suggests the possibility of an additional binding site at the entrance of the channel which when occupied blocks calcium entry. Zn 2+ has been previously shown to alter movement of ions through other channels. Gilly and Armstrong [7, 8] showed that Zn 2+ changed the opening and closing kinetics of sodium and potassium channels, probably by binding to a specific site external to these channels. In their experiments [7] Zn 2+ slowed the rate of opening of the voltage-dependent sodium channel in squid axon without changing the closing properties. They also showed [8] that Zn 2+ and Hg 2+ slowed the opening of the potassium channel and accelerated its closing at higher doses. The authors concluded that Zn 2+ binds to specific negatively charged sites associated with the opening and closing apparatus. The distribution of these binding sites is different at different channels, and this may explain why not all channels are affected in the same way. A somewhat similar alteration of kinetics by Zn 2+ was described by Mayer and Sugiyama [14] at the 'A'-channel, which conducts the transients potassium current in sensory rat neurons. We cannot exclude the possibility that Zn 2+ has a direct channel-blocking action, competing with Ca 2+ for a binding site within the channel. However, any actions of Zn 2+ within the channel cannot be very strong. Otherwise we would need much lower concentrations of Zn 2+ to block the channel, as previously shown to be the case of Pb 2+ blockade of calcium currents in this preparation [3]. The changes in the current-voltage relation and in the kinetics of activation and inactivation indicate effects at fixed charge sites, while the blockage of Ca 2+ currents by Zn 2+ can be satisfactorily explained without invoking an action within the calcium channel.
122 This research was supported by NIH Grant ES05203 to D.O.C. 1 Aniksztejn, L., Charton, G. and Ben-Aft, Y., Selective release of endogenous zinc from the hippocampal mossy fibers in situ, Brain Res., 404 (1987) 58-64. 2 Bettger, W.J. and O'Dell, B.L., A critical role of zinc in the structure and function of biomembranes, Life Sci., 28 (198l) 1425-1438. 3 Biisselberg, D., Evans, M.L., Rahmann, H. and Carpenter, D.O., Lead inhibits the voltage activated calcium current of Aplysia neurons, Toxicol. Lett., 51 (1990) 51-57. 4 Byerly, L. and Moody, W.J., Intracellular calcium ions and calcium currents in perfused neurones of the snail, Lymnea stagnalis, J. Physiol., 351 (1984) 199-216. 5 Chad, J.E. and Eckert, R., An enzymatic mechanism for calcium current inactivation in dialyzed Helix neurons, J. Physiol. 378 (1986) 31-51. 6 Chesnoy-Marchais, D., A CI - conductance activated by hyperpolarisation in Aplysia neurons, Nature, 229 (1982) 359-361. 7 Gilly, W.M.F. and Armstrong, C.M., Slowing of sodium channel opening kinetics in squid axon by extracellular zinc, J. Gen. Physiol., 79 (1982) 935-964. 8 Gilly, W.M.F. and Armstrong, C.M., Divalent cations and the activation kinetics of potassium channels in squid giant axons, J. Gen. Physiol., 79 (1982) 965496. 9 Hagiwara, S. and Byerly, L., Calcium channel, Annu. Rev. Neurosci., 4 (1981) 69-125. 10 Hermann, A. and Gorman, A.L.F., Effects of tetraethylammonium on potassium currents in a molluscan neuron, J. Gen. Physiol., 78 (1981) 87-110. I 1 Hori, N., Galeno, T. and Carpenter, D.O., Responses of pyriform cortex neurons to excitatory amino acids: voltage dependence, conductance changes and effects of divalent cations, Cell Mol. Neurobiol., 7 (1987) 73-90. 12 Kass, R.S. and Krafte, D.S., Negative surface charge density near heart calcium channels. Relevance to block by dihydropyridines, J. Gen. Physiol., 89 (1987) 629-644. 13 Kostyuk, P.G.S., Mironov, S.L. and Shuba, Ya.N., Two ion selecting filters in the calcium channel of the somatic membrane of mollusc neurons, J. Memb. Biol., 70 (1983) 83-93. 14 Mayer, M.L. and Sugiyama, K., A modulatory action of divalent cations on transient outward current in cultured rat sensory neurons, J. Physiol., 396 (1988) 417-433. 15 Nishimura, M., Zn 2+ stimulates spontaneous transmitter release at mouse neuromuscular junctions, Br. J. Pharmacol., 93 (1988) 430-436. 16 Peters, S., Koh, J. and Choi, D.W., Zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons, Science, 236 (1987) 589 593. 17 Slater, N.T., Hall, A.F. and Carpenter, D.O., Kinetic properties of cholinergic desensitization in Aplysia neurons, Proc. R. Soc. Lond., B, 223 (1984) 63 78. 18 Sloviter, R.S., A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation, Brain Res., 330 (1985) 150-153. 19 Smart, T.G. and Constanti, A., A novel effect of zinc on the lobster muscle GABA receptor, Proc. R. Soc. Lond. B, 215 (1982) 327 341. 20 Yokoyama, M., Koh, J. and Choi, D.W., Brief exposure to zinc is toxic to cortical neurons, Neurosci. Lett., 71 (1986) 351 355. 21 Zucker, R.S., Tetraethylammonium contains an impurity which alkalizes cytoplasm and reduces calcium buffer in neurons, Brain Res., 208 (1981) 473-478.