Zinc ion (Zn²⁺) is the most abundant transition metal ion in biological organisms, with mammalian cells containing an estimated 100~500 μM of total Zn²⁺ [1, 2]. Zn²⁺ is highly enriched at many, but not all, glutamatergic nerve terminals. In the brain, Zn²⁺ is released upon neuronal activity and is crucial for the control of physiological and pathophysiological brain functions. Zn²⁺ enables the modulation of neuronal excitability in the substantia nigra pars compacta (SNc) in two ways—excitation or inhibition—by altering the gating properties of voltage-gated transient A-type K⁺ channels [3]. Noh et al. proposed that Zn²⁺ increases the bursting activity of SNc neurons in a hyperpolarized state through the reduction of the transient outward K⁺ current (I_A) and decreases its tonic activity in depolarized states when I_A is increased. However, this proposal does not support previous observations that in depolarized states, Zn²⁺ enhances the firing activity of dopaminergic neurons that are acutely isolated from the rat SNc [4]. These contradictory Zn²⁺-mediated effects on excitability in SNc neurons at depolarized states were the result of investigations employing different intracellular Ca²⁺ chelators, namely, ethylene glycol tetraacetic acid (EGTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Therefore, it is important to determine whether the Zn²⁺-elicited alteration of firing activity is dependent on the Ca²⁺-buffering capacity of neurons, and on the cytosolic Ca²⁺-dependent signaling processes.

In central nervous system neurons and many other cell types, transient elevations in intracellular Ca²⁺ trigger a wide variety of Ca²⁺-dependent signaling events and processes including the regulation of firing activities and patterns. The Ca²⁺ signals are precisely restricted to local spatiotemporal domains, which are generated by a variety of different Ca²⁺ buffer systems that limit the diffusion of Ca²⁺ ions [5]. Ca²⁺ buffers—a class of cytosolic Ca²⁺-binding proteins—act as modulators of short-lived intracellular Ca²⁺ signals. Each neuron is equipped with proteins such as Ca²⁺ channels, transporters, pumps, and Ca²⁺ buffers that together shape the intracellular Ca²⁺ signals. A recent study demonstrated that the absence of Ca²⁺-binding proteins, such as parvalbumins, calbindins, and calretinins, leads to an increase in neuronal excitability through a reduction in cytosolic Ca²⁺-buffering capacity, suggesting a link between the expression of cellular Ca²⁺-binding proteins and excitability [6–8]. Alterations in the Ca²⁺-calmodulin kinase systems have been associated with some strains of seizure-susceptible mice, suggesting that calmodulin-mediated processes may play a role in the development of altered neuronal excitability and in some forms of seizure disorders [9].

Bouts of action potentials can lead to an increase in the cytosolic Ca²⁺ concentration of neurons through the activation of voltage-gated Ca²⁺ channels (VGCCs) or Ca²⁺-permeable transient receptor potential (TRP) channels, as well as through the release of Ca²⁺ from intracellular stores via Ca²⁺-induced Ca²⁺ release mechanisms. As a result, increased cytosolic Ca²⁺ levels would control neuronal excitability by modulating many ion channels, including Ca²⁺-activated K⁺ (K_Ca) channels or Ca²⁺-activated TRP channels, which regulate the firing activities and patterns of SNc dopamine neurons [10, 11]. In midbrain dopaminergic neurons, the steep Ca²⁺ concentration gradient enables it to cross the plasma membrane into neurons through open pores such as L-type VGCCs, which appear to play a major role in regulating firing activities and intracellular Ca²⁺ concentration dynamics [12, 13]. Ca²⁺ is also able to flow into the cytoplasm though the inositol trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) ion channels in the ER membrane [14]. The influx of Ca²⁺ via VGCCs or IP₃R/RyR in a depolarized state leads to the activation of small-conductance K_Ca (SK) channels. This generates a large afterhyperpolarization that affects the interspike interval of pacemaker firing in the dopaminergic midbrain neurons [15]. It was recently reported that many types of TRP channels are widespread in midbrain dopamine neurons [16, 17] and that they play a critical role in Ca²⁺ homeostasis and the regulation of firing in dopaminergic SNc neurons [10].

In light of these recent reports, this study was undertaken to examine how Zn²⁺ contributes to altered neuronal excitability in dopaminergic SNc neurons, depending on the cytosolic Ca²⁺ capacity and Ca²⁺-related channel processes, using a whole-cell patch recording in SNc neurons. We found that SK channel activity, which is influenced by changes in the cytosolic Ca²⁺ concentration under regulation by the Ca²⁺-buffering capacity, was essential in the Zn²⁺-mediated alteration of firing activity.

Brain preparation

All experiments followed the guidelines issued by Ewha Womans University on the ethical use of animals for experimentation.

Electrophysiological recordings

Solutions

For the current-clamp recordings of slices, a modified ACSF, in which NaH₂PO₄ was omitted and MgSO₄ was replaced with MgCl₂, was used as an extracellular solution to avoid any Zn²⁺-related precipitates. For the measurement of voltage-gated Ca²⁺ channels in a voltage-clamp mode, the composition of bath solution in control was (in mM): 155 tetraethylammonium chloride, 10 BaCl₂, and 10 HEPES (pH 7.4). Bath solutions were added to the recording chamber, using a perfusion system that consisted of 5 polyethylene tubes arranged in parallel in one plane for rapid exchange. All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) and Tocris (Ellisville, MO, USA). For inside-out patch single-channel recordings of SK channels, bath and pipettes contained (mM): 140 KCl, 10 HEPES, 5 MgCl₂, pH 7.2. To activate SK channels, free calcium concentration in the bath solution was adjusted by addition of 100 μM CaCl₂.

Statistical analysis

Data are presented as mean±standard error mean (S.E.M). Statistical significance was estimated with two-tailed Student’s t -tests for comparisons between two groups and was evaluated by using one-way repeated measures ANOVA followed by post hoc Tukey’s multiple comparison test and Dunnett’s post-hoc multiple comparison test for control.

Differential modulation of the excitability in SNc dopaminergic neurons by zinc, depending on internal calcium chelators

Dopaminergic SNc neurons recorded in juvenile rat brain slices were identified in a whole-cell patch-clamp by the presence of a prominent sag during hyperpolarizing current pulses, and repetitive firing due to anode break excitation, which are well-known criteria for identifying dopaminergic neurons in the SNc [4, 18, 19]. We had previously reported seemingly contradictory Zn²⁺-mediated effects on the excitability of SNc neurons, where Zn²⁺ increased the excitability of dopaminergic neurons acutely isolated from the rat SNc [4], but decreased the firing activity in the dopaminergic neurons of SNc slices [3].

To investigate the influence of the Ca²⁺-buffering capacity on Zn²⁺-mediated changes in the excitability of SNc neurons, we examined the effects of Zn²⁺ on the activity patterns of SNc neurons that had received depolarizing pulses through EGTA- or BAPTA-containing intracellular solutions in a pipette. When the pipette contained EGTA, the evoked action potentials revealed regularity in the spike frequency patterns ( A, Control). Zn²⁺ (100 μM) significantly increased the spike frequency to 181% (Fig. 1C, 100 μM Zn²⁺), but this effect was not completely reversed after the washout (Fig. 1C, Wash). However, when using the same concentration of BAPTA, the pattern of firing activities evoked by depolarization showed the opposite effect, suggesting that the Ca²⁺-buffering capacity might alter the Zn²⁺-induced modulation of excitability. The appearance of spike-frequency adaptation patterns in the pipettes containing BAPTA (Fig. 1B) suggested that the spike-frequency adaptation in SNc dopaminergic neurons occurs due to the lowering of cytoplasmic Ca²⁺ levels, which may cause a decrease in the activity of SK channels [20, 21]. In experiments with BAPTA-containing pipettes, Zn²⁺ significantly decreased the spike frequency to 63% (Fig. 1C, 100 μM Zn²⁺), but the firing activity did not recover completely after washout (Fig. 1C, Wash). BAPTA is also able to chelate Zn²⁺ influxes into the cytoplasm (K_D values: Ca²⁺, 160×10⁻⁹ M; Zn²⁺, 8×10⁻⁹ M)[22]. Therefore, we examined the effects of internal Zn²⁺ on spike frequencies using pipettes containing the Zn²⁺ chelator N,N,N’,N’-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 50 μM), which has a very high affinity for Zn²⁺ (K_D=2.6×10⁻¹⁶ M), but relatively low affinities for Ca²⁺ (K_D=4.0×10⁻⁵ M) and Mg²⁺ (K_D=2.0×10⁻² M) [23]. In experiments with TPEN-loaded pipettes, the Zn²⁺-mediated increase in excitability was less pronounced than in the EGTA experiments (Fig. 1C), suggesting that the blocking of Zn²⁺-mediated potentiation of spike activity may be due to the chelation of intracellular Zn²⁺ by both BAPTA and TPEN. However, we were unable to rule out the possibility that the membrane-permeable TPEN also chelates extracellular Zn²⁺, and we could not explain why the presence of TPEN does not result in reduced activity via enhanced I_A. Therefore, we also tested the effect of extracellular Zn²⁺-mediated augmentation of excitability on Ca²⁺ channels in the cell membrane and the endoplasmic reticulum (ER).

Involvement of VGCCs and intracellular calcium stores in the zinc-mediated augmentation of excitability

In SNc neurons, bouts of action potential can increase the cytosolic Ca²⁺ concentration either through the activation of VGCCs, or through the release of Ca²⁺ from intracellular stores via Ca²⁺-induced Ca²⁺ release signaling. The abrupt change in conductance enables Ca²⁺ to enter the neuronal cytoplasm rapidly through the open pores of L-type VGCCs in SNc dopaminergic neurons [24, 25]. First, we examined the changes in excitability in the presence of Cd²⁺ and Ni²⁺ to determine whether high-threshold VGCCs and low-threshold VGCCs are involved in the observed Zn²⁺-induced increases in excitability. In the presence of Cd²⁺ (100 μM), the Zn²⁺-induced increases in firing activities were completely blocked, and similar to the effect of BAPTA, this phenomenon did not recover after Zn²⁺ washout (Fig. 2A, top ). In contrast, the presence of Ni²⁺ (100 μM) did not influence the Zn²⁺-mediated increases in excitability. These finding suggest that the Zn²⁺-induced augmentation of firing activities was mediated by high-threshold VGCCs. To determine whether L-type high-threshold VGCCs, which mainly mediate the rhythmic firing of SNc dopaminergic neurons, contribute to the Zn²⁺-mediated augmentation of firing activities, we examined the Zn²⁺-mediated changes in firing activity in the presence of the L-type VGCCs antagonist, nifedipine (50 μM) (Fig. 2A, middle ). In accordance with previous reports [10], nifedipine reduced the spike frequency by 27% (Fig. 2B, top ), and its presence completely blocked the Zn²⁺-induced augmentation of firing activities (Fig. 2B, middle ; 100 μM Zn²⁺). However, nifedipine had no effect on the Zn²⁺-mediated increase in firing activities after the Zn²⁺ washout (Fig. 2B, bottom, Wash), suggesting that L-type VGCCs are involved in the Zn²⁺-mediated increase in firing frequency.

We further examined whether the release of Ca²⁺ from intracellular stores is responsible for the Zn²⁺-induced potentiation of excitability. To deplete intracellular Ca²⁺ stores, the Ca²⁺-ATPase inhibitor thapsigargin (1 μM) was added to the internal recording solution. Thapsigargin had no significant effects on firing activities, either in the presence of Zn²⁺, or after the Zn²⁺ washout (Fig. 2B). This suggested that Ca²⁺ release from the intracellular stores is not responsible for the Zn²⁺-induced potentiation of excitability. However, because thapsigargin leads to an increase in intracellular free Ca²⁺ by potently inhibiting the ER Ca²⁺-ATPase, we also tested the channels responsible for this Ca²⁺-induced Ca²⁺ release from the ER. Two major pathways have been identified for the release of Ca²⁺ from intracellular stores: the IP₃- and ryanodine-sensitive Ca²⁺ stores [26]. To assess the potential role of IP₃Rs in the Zn²⁺-mediated increase in firing frequency, we used a membrane-permeable IP₃R antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100 μM), which caused a 25% reduction in spike frequency but did not alter the Zn²⁺-mediated enhancement of firing activities. The release of Ca²⁺ from IP₃Rs or the influx of Ca²⁺ via VGCCs could further trigger Ca²⁺-induced Ca²⁺ release from the RyRs. To examine the possible role of RyRs in Zn²⁺-mediated increases in activity, we used caffeine to activate the RyR-sensitive Ca²⁺ stores [27]. For these experiments, we used 5 mM caffeine, which exceeds the K_D of 1.5 mM for caffeine-induced Ca²⁺ release from ER stores [28]. Caffeine had no significant effects on firing activities, either in the presence of Zn²⁺, or after Zn²⁺ washout (Fig. 2B). Dantrolene, which is a selective membrane-permeable inhibitor of RyRs [29, 30], inhibited the Zn²⁺-induced increase in spike frequency, but had no effect on firing activity after the Zn²⁺ washout (Fig. 2A, bottom; Fig. 2B). These findings indicated that the release of Ca²⁺ from the RyRs is involved in the augmentation of firing frequency in the presence of Zn²⁺.

Involvement of SK channels in the zinc-mediated augmentation of excitability

BAPTA and EGTA differ considerably in their Ca²⁺ binding rate constants, with BAPTA being about 150 times faster than EGTA [31]. Therefore, BAPTA is noticeably more effective in preventing the diffusion of free Ca²⁺ away from the entrance site at the plasma membrane. To determine whether the K_Ca channels (large-conductance K_Ca channels, BK channel; small-conductance K_Ca channels, SK channel), which are controlled by local Ca²⁺ signaling domains, are involved in the Zn²⁺-mediated modulation of excitability in SNc neurons, we examined the effects of BK and SK channels on neuronal excitability in the presence of Zn²⁺.

The SNc region has low expression of BK channels [32], and shows higher expression levels of the SK3 subunit [33, 34]. In our experiment, apamin (100 nM),—a highly specific blocker of SK channels—increased the excitability in dopaminergic neurons, whereas BK channel blockers like iberiotoxin (100 nM) and paxilline (10 μM) did not induce any changes in excitability (Fig. 3B). These observations supported previous reports [32–34] that apamin-sensitive SK channels are mainly expressed in SNc neurons, while BK channels are not. To elucidate the effect of Zn²⁺ on SK channels, we investigated whether the Zn²⁺-mediated increase in firing activity could be blocked by a pretreatment of apamin. Apamin pretreatment significantly attenuated the Zn²⁺-induced augmentation of excitability, while the BK channel blockers iberiotoxin and paxilline had no effect on it (Fig. 3C). The effects of apamin pretreatment were maintained even after the washout of extracellular Zn²⁺, suggesting that Zn²⁺ could be acting on an intracellular domain of the SK channels. These results indicated that like apamin, Zn²⁺ was capable of blocking the SK channels, thus inducing an increase in excitability. To confirm the possibility of inhibition of SK channels by Zn²⁺ in SNc neurons, we performed inside-out patch single-channel recording. We found that Zn²⁺ reduced the channel open probability of SK channel, suggesting that Zn²⁺ directly blocks inside SK channels (Fig. 4).

In order to determine how the effect of Zn²⁺ is different in the presence of BAPTA versus EGTA, the aforementioned experiments were conducted with BAPTA as well. Interestingly, in the presence of nifedipine or dantrolene, neuronal activities were lower in the BAPTA condition than in the EGTA condition (Fig. 5A and 5B), and increased neuronal activities were observed only when the SK channels were blocked by apamin in the EGTA condition (Fig. 5C). Moreover, nifedipine, dantrolene, and apamin significantly decreased the neuronal activity of Zn²⁺ under the BAPTA condition. Apamin increased neuronal activity under the EGTA condition and decreased it under the BAPTA condition, in a manner identical to the actions of Zn²⁺ (Fig. 5C).

Involvement of TRPM channels in the zinc-mediated augmentation of excitability

The tonic Ca²⁺ entry pathways that maintain basal cytosolic Ca²⁺ levels consist mainly of TRP channels, and contribute to the generation of spontaneous firings [10]. Therefore, the dopaminergic neurons in which Zn²⁺ enhanced firing activities were first tested for their response to flufenamic acid (FFA), a broad-spectrum TRP channel blocker. The application of FFA (100 μM) had no effect on firing activity, but it completely prevented the Zn²⁺-induced increase in firing activity (Fig. 6), suggesting that the potentiation of firing activity by Zn²⁺ occurred via TRP channels. On application of 10 μM of SKF96365—a widely-used blocker of canonical TRP (TRPC) channels—the potentiation of firing activity by Zn²⁺ was strongly inhibited (Fig. 6), but these effects were reversed upon the washout of SKF96365. In some preparations, SKF96365 also blocks VGCCs [35]. To determine whether the antagonists for Ca²⁺-related channels directly affect VGCCs, we tested SKF96365, dantrolene, and FFA on the VGCCs of acutely isolated SNc neurons using voltage-clamp recording (Fig. 6C). VGCC currents were elicited from isolated SNc neurons held at −100 mV by applying +30 mV, which activated all the VGCCs as expected. SKF96365 (10 μM) substantially reduced the steady-state VGCC currents by 87%, and dantrolene (10 μM) reduced the steady-state VGCC currents by 9%, but FFA (50 μM) did not significantly inhibit whole-cell steady-state VGCC currents (Fig. 6C). Therefore, we concluded that the inhibition of the Zn²⁺-induced firing potentiation by SKF96365 is mainly mediated via a VGCC inhibition, not a TRPC inhibition.

Melastatin-like TRP (TRPM) channels, which are directly activated by intracellular Ca²⁺, and conduct Na⁺ ions to further depolarize cells [36], are expressed in the neurons of the SNc [37]. To examine the possibility that TRPM channels are involved in the Zn²⁺-induced potentiation of excitability in SNc neurons, we applied 9-phenanthrol, a drug that selectively blocks TRPM channels [38]. 9-phenanthrol (50 μM) completely eliminated the Zn²⁺-induced amplification of firing activity in the tested neurons. These effects were reversed upon the washout of 9-phenanthrol, suggesting that TRPM channels could play an important role in the enhancement of firing activity by Zn²⁺.

Zn²⁺ is present in high concentration in the SN region of the parkinsonian brain [39–41], and can increase or inhibit the intrinsic firing activity of rat SNc dopaminergic neurons depending on the intracellular Ca²⁺ buffering capacity, suggesting the involvement of cytosolic Ca²⁺-dependent signaling processes. To elucidate the effect of Zn²⁺ on the excitability of dopaminergic SNc neurons, this study investigated its action on cytosolic Ca²⁺-dependent processes in relation to the intracellular Ca²⁺ buffering capacity.

The prominent results of this study can be summarized as follows: (1) In the depolarized state, Zn²⁺ increases the tonic activity during intracellular dialysis with EGTA, but decreases firing activity in the presence of BAPTA. (2) The augmentation of firing activity in the presence of Zn²⁺ is blocked by the RyR blocker dantrolene, and by the VGCC antagonists nifedipine and Cd²⁺, but not by the IP₃R antagonist 2-APB. (3) The Zn²⁺-mediated potentiation of firing activity is eliminated by the blockade of TRPM channels with FFA and 9-phenanthrol. (4) Zn²⁺ directly reduced the channel open probability of SK channels. Apamin—an SK channel inhibitor—enhances the firing activity, and its effect is attenuated by intracellular dialysis with BAPTA. Apamin blocks the augmentation of firing activity not only in the presence of Zn²⁺, but also after the washout of Zn²⁺.

We propose some hypothetical mechanisms for Zn²⁺-mediated augmentation of excitability leading to Ca²⁺ signaling processes (Fig. 7). When SK channels are blocked, the firing activity of neurons can increase. Apamin is very similar in its actions compared to Zn²⁺. Apamin enhances tonic activity in the depolarized state when the cell is intracellularly dialyzed with EGTA, whereas apamin attenuates the firing activity in the presence of intracellular BAPTA (Fig. 5C). This is in agreement with a high expression of SK3 channels, but not BK channels in SNc neurons [32, 34]. Iberiotoxin and paxilline, which are BK channel antagonists, have no effect on the firing activity in SNc neurons (Fig. 3C). In the presence of apamin, the Zn²⁺-induced enhancement of firing activity is completely blocked and this blockage is sustained even after Zn²⁺ removal from the bath solution, suggesting that Zn²⁺ possibly acts on SK channels in both the extracellular and intracellular domains. In inside-out patch single-channel recording, Zn²⁺ directly reduced the SK channel open probability (Fig. 4). These results are consistent with the previous studies that Zn²⁺ has been reported to directly inhibit the activation of K_Ca channels in other preparations such as hippocampal neurons and motor neurons [42, 43]. Therefore, we suggest that Zn²⁺ enhances the firing activity of SNc neurons via the blockade of SK channels by Zn²⁺.

Cytosolic Ca²⁺ triggers a wide variety of Ca²⁺-dependent signaling events and reaction cascades, and its levels rise only for short periods of time and at spatially restricted domains. Such local Ca²⁺ signaling domains are generated by a variety of Ca²⁺ buffer systems, such as BAPTA and EGTA, which limit the diffusion of Ca²⁺ ions that have entered the cells through VGCCs or Ca²⁺-permeable channels [44, 45]. BAPTA, but not EGTA, interferes effectively with Ca²⁺ signaling processes in Ca²⁺ nanodomains, i.e., within 20~50 nm of the Ca²⁺ source, while both modulate Ca²⁺ signaling processes equally in Ca²⁺ microdomains, i.e. between 50 nm to a few hundred nm from the Ca²⁺ source [5]. The ability of Zn²⁺ to enhance the excitability in the presence of EGTA, but not of BAPTA, suggests that rapid local changes in Ca²⁺ are necessary for the augmentation of firing activity. In SNc neurons, one potential mechanism for rapid Ca²⁺ dynamics causing Zn²⁺-mediated increases in firing activity is the co-localization of VGCCs or RyRs as Ca²⁺ sources, and SK channels as Ca²⁺ sensors. If those VGCCs or RyRs and SK channels were located in Ca²⁺ nanodomains, Zn²⁺ would have no effect on neuronal excitability when using a BAPTA-loaded pipette, because BAPTA would rapidly decrease the free Ca²⁺ concentration, thus inactivating the SK channel. However, in the presence of EGTA, the reduction of SK channel activation by Zn²⁺ results in an augmentation of excitability. Because Zn²⁺ increases the excitability in experiments with an EGTA-containing, but not a BAPTA-containing, internal solution, we suggest that VGCCs or RyRs and SK channels are located in Ca²⁺ nanodomains, and Zn²⁺ possibly attenuates SK channel activity. In the BAPTA condition, due to a decrease in the levels of free Ca²⁺, Zn²⁺-induced neuronal activity would be determined by the action of KA channels (Fig. 7).

Because the Zn²⁺-mediated potentiation of firing activity in SNc neurons was completely blocked by pretreatment with nifedipine, Cd²⁺, or dantrolene (Fig. 2), we conclude that the Zn²⁺-mediated augmentation of activity requires an increase in cytosolic Ca²⁺ concentration via the activation of L-type VGCCs and RyRs. Zn²⁺ may directly block the SK channels, and/or indirectly reduce SK channel activity via a blockade of Ca²⁺ channels such as the VGCC or RyR/IP₃R, thus attenuating the cytosolic Ca²⁺ concentration. Zn²⁺ significantly reduces the VGCC currents of SN neurons [46]. A plausible explanation for the observed Zn²⁺-induced potentiation of neuronal excitability is the indirect inhibition of SK channels caused by a decrease in Ca²⁺ influx due to the inhibition of VGCCs or RyRs by Zn²⁺.

TRPM channels, which are activated by cytosolic Ca²⁺ released from IP₃Rs on the ER membrane, further depolarize the neurons by conducting Na⁺ ions, and induce a Ca²⁺ influx by activating the VGCCs. When applied extracellularly, 2-APB—a membrane-permeable blocker of intracellular IP₃Rs—augments Ca²⁺ entry through store-operated channels (SOCs) at lower concentrations (<10 μM), and blocks TRPC channels at higher concentrations (>30 μM) [47–51]. The presence of 100 μM 2-APB failed to inhibit the Zn²⁺-mediated augmentation of firing activity, suggesting that the potentiation of neuronal activity by Zn²⁺ is not mediated by IP₃Rs and TRPC channels. SKF96365, another well-known antagonist of TRPC channels, blocks SOCs and TRPC channels at concentrations of 25~100 μM [52–54], and in some preparations also blocks VGCCs [35]. SKF96365 was a potent blocker of VGCCs in this study as well (Fig. 4C). Because VGCC antagonists, such as nifedipine or Cd²⁺ (Fig. 2B), completely blocked the Zn²⁺-mediated increase in firing activity, we suggest that the prevention of Zn²⁺ action by SKF96365 is exclusively mediated by VGCC inhibition, and not by the blockade of TRPC channels. 9-phenanthrol blocks TRPM4 channels in a human embryonic kidney cell expression system with a half-maximal inhibitory concentration (IC₅₀) of 16.7 μM under conventional whole-cell patch-clamp conditions, and does not alter the activity of TRPC channels, BK channels, inward-rectifying K⁺ channels, or VGCCs [55]. Therefore, FFA and 9-phenanthrol halt the Zn²⁺-mediated potentiation of firing activity by inhibiting the TRPM—most likely, TRPM4—channels. We suggest that the Zn²⁺-mediated inhibition of TRPM channels causes a decrease in SK activity via the reduction of cytosolic Ca²⁺, which contributes to the observed augmentation of firing activity in the SNc neurons.

In summary, we propose that (1) extracellular Zn²⁺ reduces the cytosolic Ca²⁺ concentration not only via the blockade of VGCCs, but also by reducing the Ca²⁺-mediated inactivation of RyRs/TRPM channels, resulting in a decrease in the activation of SK channels, and (2) Zn²⁺ directly blocks SK channels via interactions with an intracellular domain. This suggests that in the SNc, the blockade of SK channels by Zn²⁺ directly and/or indirectly induces the Zn²⁺-mediated augmentation of excitability.

Several Ca²⁺-binding proteins—or Ca²⁺ buffering proteins, including calretinin, calbindin and parvalbumin—are found throughout the central nervous system, including in the midbrain dopaminergic neurons, and help maintain a tight regulation of Ca²⁺ signals [56–58]. These high-affinity Ca²⁺-binding proteins act as buffers to prevent abrupt changes in the intracellular Ca²⁺ concentration [59, 60], and this is essential in preventing cell dysfunction and excitotoxic damage caused by high intracytoplasmic concentrations of Ca²⁺ [49, 61]. Some reports emphasize the role of Ca²⁺-sensing receptor or Ca²⁺-binding proteins/ Ca²⁺-buffering protein in pathophysiology of glaucoma and vulnerability to traumatic noise [62, 63]. The pathology of Parkinson’s disease involves a significant loss of parvalbumin-immunoreactivity in SN neurons [64], suggesting that this Ca²⁺-buffering protein plays a crucial role in maintaining normal Ca²⁺ signaling processes and Ca²⁺ homeostasis. Based on the results our experiments using EGTA and BAPTA, we propose that Zn²⁺ could increase the excitability of SNc neurons in pathological conditions involving a decrease in the activity of Ca²⁺-binding proteins, similar to the EGTA condition, whereas Zn²⁺ could decrease the firing activity in physiological conditions involving an increased activity of Ca²⁺-binding proteins, similar to BAPTA condition. This suggests that exogenous or endogenous factors such as Zn²⁺ could differentially modulate the excitability of neurons, depending on the physiological or pathological state of synaptic as determined by the Ca²⁺-buffering capacity. By attenuating Ca²⁺ influx into the cytosol, Zn²⁺ may also compensate for the loss of SNc dopaminergic neurons during the development of Parkinson’s disease [65], thus maintaining normal information processes in the basal ganglia until a critical mass of dopaminergic neurons are generated.

The present research was supported by the research fund of Dankook university in 2017.