Metal interactions with voltage- and receptor-activated ion channels.

Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions.


Introduction
In excitable cells ion channels are directly responsible for the rapid electric signaling and are also involved in the modulation of excitability. Calcium ions play a prominent role in the modulation of excitability, because these ions permeate through various ion channels and activate K channels at the internal face of the membrane (1). In addition, internal Ca ions trigger a range of biochemical processes involved in signal transduction (2). Various metal ions may substitute for Ca. These metal ions activate or block Ca-dependent processes, which may lead to altered excitability and may disturb the intracellular Ca homeostasis, ultimately leading to cell death (3). This paper was presented at the Second International Meeting on Mechanisms of Metal Toxicity and Carcinogenicity held 10-17 January 1993 in Madonna di Campiglio, Italy.
The authors thank Ms. P. Martens for maintaining cell culture and Ing. A. de Groot for expert technical assistance. Acetylcholine receptor research has been financially supported by Shell Internationale Research Maatschappij BV, and calcium-activated potassium channel research by the Foundation for Biological In mouse neuroblastoma cells of the clone N BE-11 5, a range of distinct types of ion channels and receptors have been identified. These cells express voltage-activated sodium channels, at least two types of voltage-activated Ca channels and both voltage-activated and Ca-activated K channels (4)(5)(6)(7). Recently, serotonin 5-HT3 receptors and neuronal type nicotinic acetylcholine (ACh) receptors, which are directly coupled to distinct cation channels, have been characterized in NlE-115 cells (8,9). The detailed knowledge of the properties of ion channels in neuroblastoma cells and the suitability of these cultured cells for intracellular electrophysiology permit the separation of the various types of ion channels in voltage clamp experiments and thereby the investigation of effects of metal ions on each type of ion channel.
The results reviewed below demonstrate that metal ions interact with multiple sites on ion channels in the mammalian nervous system.

Materials and Methods
Mouse neuroblastoma cells of the clone NlE-115 (10) were grown as described previously (9). Experiments were carried out using the whole-cell voltage clamp or the single-channel patch-clamp technique (11). Fire-polished glass pipettes had an internal tip diameter of 1 to 1.5 pim and a resistance of 3 to 5 MO. Membrane cur-rents recorded under voltage clamp were low-pass filtered, digitized (8 bits; 1024 points per record) and stored on magnetic disc for off-line computer analysis.
Voltage-activated ion currents were evoked by step depolarizations of the membrane. Receptor-activated ion currents were evoked by whole-cell superfusion with external solution containing known concentrations of agonist and/or metal ions for adjustable periods (.1 sec). In between agonist-induced responses, evoked at intervals of 3-4 min, desensitization was completely reversed by continuous superfusion of the cell with external solution. Single Ca-activated K channels were recorded by the single channel patch clamp technique from inside-out membrane patches of N 1 E-1 5 cells. Patches were superfused with buffered Ca-free and with internal solutions in which Ca or other metal ions were buffered with citric acid. All experiments were carried out at room temperature (20-240C).
Different external and pipette solutions for optimum recording of the various types of ion currents independently were prepared from ultrapure chemicals and double glass-distilled water. The ionic compositions of the solutions are presented in Table 1, which includes the total contamination by Pb as calculated from the data supplied with the chemicals. Indicated metal-ion concentrations refer to the Environmental Health Perspectives

Effecms on Voltage-activated Ion Channels
In external solution containing 50 mM of Ba ions membrane depolarization from a holding potential of -80 mV to + 10 mV evokes a fast transient as well as a noninactivating inward Ba current in whole cell voltage-clamped neuroblastoma cells. These Ba current components are carried by distinct transient and sustained types of voltage-activated Ca channels (5). Superfusion with Pb2+ causes a reduction of the amplitude of both types of Ba current within 5 to 10 min and the blocking effects are reversed after 5 to 10 min of washing with control external solution.
The blocking effect of Pb2+ on the transient Ba current component is concentration-dependent with an IC50 value of 4.8 ± 0.8 pM and a slope factor of -0.88 ± 0.14 ( Figure 1). The transient and sustained Ba current components appear to have a similar sensitivity to Pb2+. In contrast, 10 to 100 pM Pb2+ does not affect voltage-activated sodium current, evoked by a step depolarization to 0 mV for 20 msec preceded by a conditioning membrane hyperpolarization to -120 mV for 100 msec (not shown).
Effets on Receptor-Activated Ion Channel Whole-cell superfusion of voltage-clamped NlE-115 cells with external solution containing acetylcholine (ACh) evokes a transient inward current. The ACh-induced inward current is mediated by neuronal type nicotinic ACh receptors, which are selectively blocked by K-bungarotoxin and Environmental Health Perspectives During superfusion with 1 pM Pb2+ the peak amplitude of the ACh-induced inward current was reduced to 18% of the control value. Note that in addition 1 pM Pb2+ delayed the decay of the ACh-induced inward current. Membrane potential was held at -80 mV. Superfusion periods are indicated by bars. Lower panel: Concentration-effect curve of block by Pb2+ of the nicotinic ACh receptor-activated ion current. Ordinate represents the inward current peak amplitude normalized to control value. The data were fitted by the sum of two sigmoidal functions, according to the equation i/imax = 1/{1+(EC%4Pb2+])n}. The estimated parameters of the fitted curve (solid line) are: IC50 = 19 ± 6.3 nM; EC50 = 21 ± 5.5 pM and the slope factors n are -0.45 ± 0.08 and 0.84 ± 0.15, respectively. The discontinuous lines represent the concentration-effect curve for the blocking effect of Pb2+ and for the reversal of block, according to the fitted parameters. Modified after Oortgiesen et al. ( 14). are insensitive to ic-bungarotoxin (9). Superfusion with Pb2+ rapidly reduces the amplitude of the 1 mM ACh-induced inward current. This effect is partially reversed during a 5 to 10 min period of washing with control external solution. Figure 2 shows that after 14 min of superfusion with 1 pM Pb2+, the peak amplitude of the ACh-induced inward current is reduced to 18% of the control value. In  Figure 2) with an IC50 value of 19 ± 6 nM for block and an EC50 of 21 ± 5 pM for the reversal of the blocking effect. The slope factors of the two curves are -0.45 ± 0.08 and 0.84 ± 0.15, respectively. Superfusion of neuroblastoma cells with 3 pM serotonin (5-HT) activates a transient inward current, mediated by an independent population of serotonin 5-HT3 receptor-activated ion channels (8).
Pb2+ reversibly reduces the amplitude of the 5-HT-induced inward current within 4 to 8 min, without changing the kinetics of the inward current. During superfusion with 1 pM Pb2+, the peak amplitude of the 5-HT-induced inward current is reduced to 74 ± 5% (n=3) of the control value. The estimated values of the IC50 and the slope factor of the concentration-effect curve of block of the 5-HT-induced inward current by Pb2+ are 49 ± 18 pM and -0.32 ± 0.04, respectively (14).

Effects on Calcium-Actvated Potassium Channels
Two types of Ca-activated K channels can be identified in excised membrane patches of N1E-1 15 neuroblastoma cells. SK channels, which have a low single-channel conductance of 5 pS, are potently blocked by the bee venom peptide apamin and show a relatively high sensitivity to Ca. BK channels, which have a high single-channel conductance of 98 pS, are sensitive to block by Volume  tetraethylammonium ions (TEA) and are less sensitive to Ca (7). Figure 3 shows representative traces of single SK and BK channel recordings from two inside-out excised patches of Ni E-115 membrane at a holding potential of 0 mV. In these experiments, SK and BK channels were maximally activated by superfusion of the inside of the patches with solutions containing 14 Mg2+. The sequences show that Pb2+ is more potent than Ca2+ in activating both SK and BK channels. Cd2+ is also a very potent activator of SK channels, but is unable to activate BK channels even at a concentration of 100 pM. Mg2+ is completely inactive at concentrations up to 100 pM (15).
Efets on Metal Ion-activated Ion Channels Superfusion with Pb2+ also induces a slow, noninactivating and reversible inward current in NlE-1 15 cells. The amplitude of this inward current increases in the range of 1 to 200 pM Pb2+. Exposure of excised outside-out membrane patches to Pb2+ revealed that the slow inward current is mediated by the opening of discrete ion channels (Figure 4a) with a single-channel conductance of 24 pS. Single-channel events can be detected at Pb2+ concentrations . 0.1 pM. Chelation of external Pb2+ by superfusion with EGTA-containing solution fully abolished single-channel activity as illustrated in Figure 4b for a patch containing multiple channels. The reversal of the whole-cell membrane current and of the single-channel currents at approximately 0 mV (not shown) suggests that the current is carried by nonselective cation channels. The Pb2+-induced membrane current appears not to be mediated by various known types of ion channels, since it can neither be blocked by external tetrodotoxin, TEA, d-tubocurarine, atropine, the potent 5-HT3 antagonist ICS 205-930, nor by internal EGTA. In N I E-1 15 neuroblastoma cells Cd and Al activate ion channels similar to those activated by Pb2+ (16).

Discussion
Evaluation of direct effects of Pb2+ on ion channels in cultured N 1 E-1 15 neuroblastoma cells demonstrates differential sensitivities of various types of receptor activated and voltage-activated ion channels to this heavy metal. The results, summarized in Table 2, show that the neuronal nicotinic receptor-activated ion current is the more sensitive target, and that it is selectively blocked by nanomolar concentrations of Pb2+. Inhibitory as well as acti- Within the class of receptor-activated ion channels Pb2+ selectively affects the neuronal nicotinic receptor, because the serotonin 5-HT3 receptor-activated ion current is affected only by Pb2+ at micromolar concentrations. In addition, glutamate NMDA receptor-activated ion channels in rat hippocampal neurones are also blocked by Pb + only at concentrations in the high micromolar range (17).
Voltage-activated Ca channels in NIE-  (19,20). The results show that, despite the close relation between voltage-activated sodium and Ca channels (21) (22,23,(28)(29)(30). A continuous spontaneous release of low amounts of neurotransmitters and block of nerve-evoked neurotransmitter release may disturb neural networks particularly during development (31). SK and BK channels, two distinct types of Ca-activated K channels of N1E-115, are directly activated by intracellular Pb2+ applied to excised membrane patches. Voltage-activated K channels have been reported to be insensitive to Pb2+ (19,20).
In particular, the SK channel appears sensitive to submicromolar Pb2+ concentrations. The SK channel is responsible for the afterhyperpolarization that follows the action potential (6) and is involved in the regulation of neuronal firing fre%uency. Activation of SK channels by Pb + may cause hyperpolarization, increase the excitation threshold and reduce action potential duration. These effects would contribute to a reduction of neurotransmitter release when occurring in the presynaptic terminal. Of various metal ions, Pb + is the more potent to activate SK and BK channels, whereas Cd2+ is a potent activator of SK channels, but does not activate BK channels at concentrations below 100 pM. The distinct potency sequences for activation of subtypes of Caactivated K channels deviate from the potency sequence to block voltage-activated Ca channels in N1 E-1 15 cells (5).
Although Pb2+ is also the more potent Ca channel blocker, Cd2+ blocks sustained Ca current more potently than Co2+ and the two metal ions are equipotent in blocking transient Ca current (5). This suggests that metal ions interact with ion channel proteins in a highly selective manner.
At high concentrations, external Pb2+ directly activates a slow inward current in N1 E-1 15 cells. Results of experiments with channel blockers, receptor antagonists, and chelated internal Ca indicate that this slow inward current is not mediated by a previously described type of neurotransmitter receptor-activated ion channel, voltage-activated ion channel or Ca-activated ion channel (16). The comparison of effects on subsets of ion channels that are functionally and structurally related shows that Pb2+ selectively interacts with specific membrane proteins. In addition, different metal ions selectively modify distinct target sites. This implies that for any particular neuron the effects of metal ions on electrical activity may vary, depending on metal ion species, the extra-and intracellular concentrations, and on the presence, availability, and density of specific types of ion channels.