Subunit-specific modulation of T-type calcium channels by zinc

Abstract

Zinc (Zn²⁺) functions as a signalling molecule in the nervous system and modulates many ionic channels. In this study, we have explored the effects of Zn²⁺ on recombinant T-type calcium channels (Ca_V3.1, Ca_V3.2 and Ca_V3.3). Using tsA-201 cells, we demonstrate that Ca_V3.2 current (IC₅₀, 0.8 μm) is significantly more sensitive to Zn²⁺ than are Ca_V3.1 and Ca_V3.3 currents (IC₅₀, 80 μm and ∼160 μm, respectively). This inhibition of Ca_V3 currents is associated with a shift to more negative membrane potentials of both steady-state inactivation for Ca_V3.1, Ca_V3.2 and Ca_V3.3 and steady-state activation for Ca_V3.1 and Ca_V3.3 currents. We also document changes in kinetics, especially a significant slowing of the inactivation kinetics for Ca_V3.1 and Ca_V3.3, but not for Ca_V3.2 currents. Notably, deactivation kinetics are significantly slowed for Ca_V3.3 current (∼100-fold), but not for Ca_V3.1 and Ca_V3.2 currents. Consequently, application of Zn²⁺ results in a significant increase in Ca_V3.3 current in action potential clamp experiments, while Ca_V3.1 and Ca_V3.2 currents are significantly reduced. In neuroblastoma NG 108-15 cells, the duration of Ca_V3.3-mediated action potentials is increased upon Zn²⁺ application, indicating further that Zn²⁺ behaves as a Ca_V3.3 channel opener. These results demonstrate that Zn²⁺ exhibits differential modulatory effects on T-type calcium channels, which may partly explain the complex features of Zn²⁺ modulation of the neuronal excitability in normal and disease states.

Zinc (Zn²⁺) is involved in many biochemical and physiological processes in mammalian tissues, being an essential micronutrient (Sandstead, 2000), a structural component of many proteins (Berg, 1990) and a signalling molecule (Weiss et al. 2000). Although Zn²⁺ is mostly bound to proteins in biological systems, it is implicated in the regulation of neuronal excitability (Smart et al. 1994) and synaptic plasticity (Li et al. 2001), as well as in many neurodegenerative diseases, like Alzheimer's disease (Bush et al. 1994; Ritchie et al. 2003), amyotrophic lateral sclerosis (Estevez et al. 1999) and Parkinson's disease (Forsleff et al. 1999). Its involvement in some forms of epilepsy is also reported (Pei & Koyama, 1986), but remains unclear. In the brain, high concentrations are found especially in forebrain regions, including the hippocampus, amygdala and neocortex, as well as in the grey matter. The highest concentration (approaching 300 μm) is found in the mossy fibre terminals of the hippocampus (Frederickson et al. 1983, 2005). Zn²⁺ is selectively stored in, and released from, the presynaptic vesicles of mostly glutamatergic neurons, which are found chiefly in the mammalian cerebral cortex (Assaf & Chung, 1984; Howell et al. 1984; Hartter & Barnea, 1988). Such pools of Zn²⁺ can be released following membrane depolarization or neural activity in a calcium-dependent manner (Huang, 1997; Lin et al. 2001).

Free Zn²⁺ modulates many membrane receptors, transporters and channels (reviewed in Mathie et al. 2006), such as NMDA and AMPA (Paoletti et al. 1997; Shen & Yang, 1999), ASIC (Baron et al. 2002), GABA_A (Casagrande et al. 2003), serotonin (Hubbard & Lummis, 2000), glycine (Chattipakorn & McMahon, 2002) and P2X receptors (Xiong et al. 1999). Zn²⁺ also regulates several voltage-gated ionic conductances, including K⁺ (Easaw et al. 1999; Zhang et al. 2001; Teisseyre & Mozrzymas, 2002; Gruss et al. 2004; Clarke et al. 2004; Prost et al. 2004; Kim et al. 2005), Na⁺ (White et al. 1993; Amuzescu et al. 2003; Kuo et al. 2004) and Ca²⁺ conductances (Magistretti et al. 2003; Noh & Chung, 2003).

Several lines of evidence indicate that LVA/T-type Ca²⁺ channels, especially in neurons, are inhibited by micromolar concentrations of Zn²⁺ (Busselberg et al. 1992; Todorovic & Lingle, 1998; Nikonenko et al. 2005). T-type Ca²⁺ channels correspond to a subfamily of voltage-gated Ca²⁺ channels with specific hallmarks: low-voltage activation, transient inactivation kinetics and small unitary conductance (reviewed in Perez-Reyes, 2003). T-type Ca²⁺ channels are involved in cardiac pacemaker activity (Hagiwara et al. 1988; Mangoni et al. 2006), neuronal firing activity (Huguenard, 1996), sleep (Anderson et al. 2005), hormone secretion (Chen et al. 1999; Leuranguer et al. 2000) and fertilization (Arnoult et al. 1996). T-type Ca²⁺ channels are also implicated in the pathogenesis of epilepsy (Tsakiridou et al. 1995; Kim et al. 2001; Zhang et al. 2002), pain (Todorovic et al. 2002; Kim et al. 2003; Bourinet et al. 2005) and cardiac hypertrophy (Nuss & Houser, 1993). T-type Ca²⁺ channels were cloned only recently (Ca_V3.1/α_1G, Ca_V3.2/α_1H and Ca_V3.3/α_1I; reviewed in Perez-Reyes, 2003) and their specific pharmacological properties have not yet been clearly specified. Because Zn²⁺ is a bioactive molecule, its selectivity and inhibitory mechanism with respect to Ca_V3.1, Ca_V3.2 and Ca_V3.3 channels is in need of detailed investigation. Given that the three Ca_V3 isotypes exhibit differences in their biophysical properties (Chemin et al. 2002), as well as distinct neuronal distributions (Talley et al. 1999), Zn²⁺ modulation of Ca_V3 channels might differentially modulate neuronal excitability. In the present study, we provide a detailed analysis of the complex modulatory effects of Zn²⁺ on the human recombinant Ca_V3.1, Ca_V3.2 and Ca_V3.3 channels.

Methods

Cell culture and transfection protocols

HEK-293/tsA-201 cells were cultivated in Dulbecco's modified Eagle's medium (Laboratoires Eurobio, Courtaboeuf, France) supplemented with glutamax and 10% fetal bovine serum (Life technologies) using standard techniques. Transfection was performed using jet-PEI (QBiogen, Illkirch, France) with a DNA mix containing 10% of a green fluorescent protein (GFP) plasmid and 90% of either of the pCDNA3 plasmid constructs that code for human Ca_V3.2 subunit (Cribbs et al. 1998) and human Ca_V3.3 subunit (Monteil et al. 2000b; Gomora et al. 2002). Studies on the human Ca_V3.1 subunit was performed either using transient transfection of the Ca_V3.1 subunit (Monteil et al. 2000a) or a clonal CHO-Ca_V3.1 cell line (Traboulsie et al. 2006). This CHO cell line was cultured in α-MEM (EuroBio, Les Ulis, France) supplemented with the antibiotic (Life Technologies, Cergy Pontoise, France) G418 at 300 μg ml⁻¹. Neuroblastoma NG 108-15 cells were cultured and transfected with the Ca_V3.3 construct as described earlier (Chemin et al. 2001; Chevalier et al. 2006).

Electrophysiology recordings and data analysis

Electrophysiological recordings were performed 2–5 days after transfection, as previously described (Chemin et al. 2002). Extracellular solution contains (mm): 2 CaCl₂, 145 TEA-Cl and 10 Hepes (pH to 7.4 with TEA-OH). Pipettes have a resistance of 2–3 MΩ when filled with a solution containing (mm): 110 CsCl, 10 EGTA, 10 Hepes, 3 Mg-ATP and 0.6 GTP (pH to 7.2 with CsOH). Currents were recorded using an Axopatch 200 and pCLAMP data acquisition software (Axon Instruments, Inc., Union City, CA, USA). The sampling frequency for acquisition was 10 kHz and data were filtered at 2 kHz. For action potential clamp studies, a thalamo-cortical relay cell firing activity generated by the NEURON model (Hines & Carnevale, 1997) was used as previously described (Chemin et al. 2002). Zinc chloride (ZnCl₂) was purchased from Sigma-Aldrich (France), and was dissolved in extracellular medium at 1 m as a stock solution, kept at 4°C and applied at final concentrations to recorded cells by a gravity-driven perfusion device controlled by solenoid valves.

Current–voltage curves (I–V curves) were fitted using a combined Boltzmann and linear ohmic relationships, where:

To minimize the consequence of current rectification near reversal potential on the determination of conductance, the current values greater than +30 mV were not considered for the fit. The normalized conductance–voltage curves were fitted with a Boltzmann equation:

Similarly, steady-state inactivation curves were fitted using:

The dose–response curves were fitted using a sigmoidal dose–response function:

Current clamp experiments in NG 108-15 cells were performed as described earlier (Chevalier et al. 2006). Cells were bathed in (mm): 130 NaCl, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 11 glucose, 10 Hepes (pH 7.4 with NaOH). The pipette solution contained (mm): 5 NaCl, 50 KCl, 65 K₂SO₄, MgCl₂, 2 ATP and 10 Hepes (pH 7.3 with KOH). Student's t test was used to compare the different values, and differences were considered significant at P < 0.05. Results are presented as the mean ± s.e.m., and n is the number of cells used.

Results

Zn²⁺ inhibition of cloned T-type calcium channels

To determine the effect of Zn²⁺ on cloned T-type Ca²⁺ channels, macroscopic Ca²⁺ current was recorded in the whole-cell configuration in transiently Ca_V3 transfected HEK-293/tsA-201 cells. Figure 1 shows the effect of 1 μm Zn²⁺ on cloned Ca_V3.1 (Fig. 1A), Ca_V3.2 (Fig. 1B), and Ca_V3.3 (Fig. 1C) T-type Ca²⁺ channels. At 1 μm, Zn²⁺ significantly inhibited Ca_V3.2 (by 55%), but not Ca_V3.1 and Ca_V3.3 currents, indicating that Zn²⁺ was selective for Ca_V3.2 at a low concentration (Fig. 1D). The block by Zn²⁺ was fast and fully reversed upon washout (Fig. 1E). The dose–response relationships of Zn²⁺ inhibition (Fig. 1F) were then measured showing that Ca_V3.2 is highly sensitive to Zn²⁺ (IC₅₀ = 0.78 ± 0.07 μm, n = 11) whereas the IC₅₀ values determined for Ca_V3.1 were 100-fold higher (IC₅₀ = 81.7 ± 9.1 μm, n = 7), and 200-fold higher for Ca_V3.3 currents (IC₅₀ = 158.6 ± 13.2 μm, n = 6). The Hill coefficients were 0.7 ± 0.1 for Ca_V3.1; 0.5 ± 0.1 for Ca_V3.2 and 0.7 ± 0.1 for Ca_V3.3.

Figure 1. Zinc inhibits human Ca_V3 T-type Ca²⁺ channels.

A–D, effect of 1 μm Zn²⁺ on Ca_V3.1 (A), Ca_V3.2 (B) and Ca_V3.3 (C) Ca²⁺ currents in the presence of 2 mm Ca²⁺. Currents were elicited by −30 mV test pulse (TP) applied from a HP of −100 mV. D, bar graph of the average inhibition of Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents by 1 μm Zn²⁺. E, time course of Ca_V3.2 peak current amplitude (measured for a TP at −30 mV from a HP of −100 mV) recorded before, during and after exposure to 1 μm Zn²⁺. F, dose–response relationships for Zn²⁺ inhibition of Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents. Fraction of unblocked peak current (I/I_Ctrl) is plotted against Zn²⁺ concentration. The IC₅₀ values were obtained from fitted data using a sigmoidal dose–response with variable Hill slope equation.

T-current inhibition by Zn²⁺ is state dependent

Current-voltage relationships (I–V curves) were recorded for the three cloned T-type Ca²⁺ channels at Zn²⁺ concentration near IC₅₀ values, as illustrated in Fig. 2 for Ca_V3.2 in control condition (Ctrl; Fig. 2A) and after 1 μm Zn²⁺ (Fig. 2B), as well as for Ca_V3.3 in control condition (Ctrl; Fig. 2C) and after 100 μm Zn²⁺ (Fig. 2D). In the presence of 1 μm Zn²⁺, while about half of the Ca_V3.2 inward current was blocked (51 ± 7%, n = 5), the Ca_V3.2 outward current (at +70 mV) was reduced by only 14 ± 6% (n = 5), as illustrated in Fig. 2E. In the presence of 100 μm Zn²⁺, which reduced by 45 ± 7% (n = 6) Ca_V3.3 inward current, inhibition of the Ca_V3.3 outward current was 24 ± 11% (Fig. 2F). These data indicate a distinct ability of Zn²⁺ to act as a pore channel blocker on Ca_V3.2 and Ca_V3.3 channels. The effects of Zn²⁺ on activation properties were then determined from I–V curves. Notably, Zn²⁺ induced a significant decrease of the current amplitude at potentials positive to −50 mV (Fig. 3A–C). Except for Ca_V3.2, Zn²⁺ (Fig. 3C) produced an apparent −10 mV shift in Ca_V3.3 current activation (V_0.5 = −45.5 ± 0.6 mV, n = 8 in control; and −55.2 ± 0.4 mV, n = 8 upon 100 μm Zn²⁺ treatment) and −6 mV in Ca_V3.1 current activation (V_0.5 = −43.9 ± 1.0 mV, n = 10 in control; and −49.3 ± 1.4 mV, n = 10 upon 100 μm Zn²⁺ treatment). These results indicate that Zn²⁺ inhibition is complex, and unlikely to involve only a pore block that would induce a positive shift in the I–V curve, but to involve also an allosteric modulation that affects channel activation.

Figure 2. Family of control and zinc-inhibited Ca_V3.2 and Ca_V3.3 currents.

A and B, typical family of Ca_V3.2 currents in control (A) and after application of 1 μm Zn²⁺ (B). C and D, typical family of Ca_V3.3 currents in control (C) and after application of 100 μm Zn²⁺ (D). The selected current traces correspond to the following depolarizing voltage pulses (mV): −80, −70, −60, −55, −50, −45, −40, −30 (peak current), −20, 0, +30, +60 and +90, from a HP of −100 mV. E and F, superimposed current traces obtained for depolarizing pulses to −30 mV at +70 mV in control and after Zn²⁺ application for Ca_V3.2 currents (E) and Ca_V3.3 currents (F). The percentage of Ca_V3.2 and Ca_V3.3 current inhibition is presented as insets in panels E and F for pulses at −30 mV (inward current, white bars) and +70 mV (outward current, black bars).

Figure 3. Effect of zinc on current–voltage relationships, steady-state activation and inactivation of Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents.

A–C, mean current–voltage (I–V) relationships of Ca_V3 currents in the absence (filled symbols) or presence (open symbols) of Zn²⁺. The current amplitude was normalized to the maximum amplitude (in control condition) for each cell. Currents were recorded from a HP of −100 mV, stepping from −80 mV to +30 mV, once every 10 s. D–F, steady-state activation curves were obtained from the fitting of the I–V curves. Steady-state inactivation for Ca_V3.1 (D), Ca_V3.2 (E) and Ca_V3.3 (F) currents in the absence (filled symbols) and the presence of Zn²⁺ (open symbols). The steady-state inactivation was estimated from the variation of the current amplitude at −30 mV after a 5 s conditioning pulse of increasing amplitude (5 mV increment) from −110 mV to −40 mV. The normalized peak current amplitude was plotted as a function of the conditioning pulse.

The steady-state inactivation properties were analysed in the presence of Zn²⁺ and the voltage of half-maximal inactivation (V_0.5-inac) was determined. Figure 3D–F shows that Zn²⁺ shifted the inactivation curves to more negative potentials. The V_0.5-inac values were −72.5 ± 1.4 mV (control) and −79.4 ± 0.6 mV (100 μm Zn²⁺) for Ca_V3.1 (n = 10); −72.3 ± 0.9 mV (control) and −77.8 ± 0.9 mV (1 μm Zn²⁺) for Ca_V3.2 (n = 9); and −72.4 ± 1.8 mV (control) and −79.4 ± 0.6 mV (100 μm Zn²⁺) for Ca_V3.3 (n = 10). To evaluate Zn²⁺ binding to inactivated T-channels we have measured the current inhibition at various holding potentiels (HPs). Ca_V3.1 current inhibition in the presence of 100 μm Zn²⁺ was more important at −70 mV (77 ± 3% inhibition, n = 5), compared to the inhibition measured from a HP of −100 mV (48 ± 5% inhibition), suggesting that Zn²⁺ possibly binds on inactivated T-channels. In addition, Zn²⁺ inhibited Ca_V3.1 currents by 49 ± 4% in the absence of stimulation (n = 5, HP of −100 mV), indicating that inhibition of T-current by Zn²⁺ is not use dependent.

Differential effects of Zn²⁺ on Ca_V3 current kinetics

Figure 4 shows that Zn²⁺ differentially regulated activation and inactivation kinetics of cloned T-channels. Zn²⁺ (100 μm) significantly accelerated Ca_V3.1 activation kinetics (at −40 mV, τ = 5.3 ± 0.3 ms and 3.3 ± 0.2 ms, n = 11, for control and after Zn²⁺ application, respectively; Fig. 4Aa and b). In contrast, 100 μm Zn²⁺ significantly slowed the inactivation kinetics of the Ca_V3.1 current (14.7 ± 1.2 ms and 62.6 ± 13.0 ms, n = 11 for control and after Zn²⁺ application, respectively; Fig. 4Ac). By contrast, no change in Ca_V3.2 current kinetics was observed upon Zn²⁺ treatment (Fig. 4B), while only Ca_V3.3 inactivation kinetics was slowed down (τ = 129.6 ± 9.4 ms and 194.6 ± 9.5 ms, n = 9 at −40 mV, in control and after 100 μm Zn²⁺ application; Fig. 4C). To study the possible effects of Zn²⁺ on the deactivation kinetics, tail currents were recorded and the time constant of current decay (τ_decay) was determined. Figure 5 shows that Zn²⁺ markedly slowed down the deactivation kinetics of Ca_V3.3 current. At −80 mV, as illustrated in Fig. 5A, deactivation kinetics of Ca_V3.3 current varied from τ = 1.7 ± 0.2 ms (control) to τ = 49.3 ± 3.4 ms (100 μm Zn²⁺, n = 5; Fig. 5D), whereas no significant change was observed for Ca_V3.1 (n = 13) and Ca_V3.2 (n = 7) deactivation currents above. Slowing of Ca_V3.3 current deactivation kinetics was observed for a wide range of membrane potentials (Fig. 5A, inset). Altogether, these data indicate that Zn²⁺ selectively modifies the gating of cloned Ca_V3 T-type Ca²⁺ channels.

Figure 4. Effects of zinc on activation and inactivation kinetics of Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents.

Aa–Ca, original Ca_V3.1, Ca_V3.2 and Ca_V3.3 current traces at −40 mV from a HP of −100 mV before (filled symbols) and after (open symbols) Zn²⁺ application (100 μm for Ca_V3.1 and Ca_V3.3 currents; 1 μm for Ca_V3.2 current). Current traces were normalized in amplitude in order to visualize the kinetics modifications. Ab–Cb, time constants of activation kinetics plotted as a function of membrane potential (V_m) for Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents before (filled symbols) and after (open symbols) Zn²⁺ application (100 μm for Ca_V3.1 and Ca_V3.3 currents; 1 μm for Ca_V3.2 current). Ac–Cc, time constants of inactivation kinetics, as in Ab–Cb. Statistically significant differences are indicated on the plots.

Figure 5. Effects of zinc on the deactivating tail currents of Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents.

A–C, examples of normalized tail currents at −80 mV elicited after a −30 mV TP from a HP of −100 mV (filled symbols for control traces and open symbols (bold traces) in the presence of Zn²⁺). For each Ca_V3 subunit, the TP duration was adjusted in order to trigger maximum deactivating currents (4 ms for Ca_V3.1, 7 ms for Ca_V3.2, and 28 ms for Ca_V3.3 currents). The decay of tail currents was fitted using a single exponential equation. The insets show the voltage dependence of Ca_V3 current deactivation from −120 mV to −60 mV. D, bar graph of the mean time constant of the tail current decay (at −80 mV) under control conditions (filled) and in the presence of Zn²⁺ (open).

Differential Zn²⁺ modulation of Ca_V3 channels in action potential clamp experiments

We have used a thalamo-cortical relay (rTC) firing activity generated by the NEURON model (Chemin et al. 2002) as a voltage command to better visualize the multiple effects of Zn²⁺ on Ca_V3 channel gating. This voltage command was used in action potential clamp experiments on the three cloned T-type Ca²⁺ channels expressed in tsA201 cells (Fig. 6). Using reticular and relay thalamic neuron activities as voltage commands, we previously showed that Ca_V3.3 channels produced a sustained Ca²⁺ current due to slow activation and inactivation kinetics, while Ca_V3.1 and Ca_V3.2 currents generated more transient inward Ca²⁺ current in these experimental conditions (Chemin et al. 2002). Each single action potential of the rTC train induces an inward Ca²⁺ current that reflects instantaneous inactivation and deactivation properties of Ca_V3.3 channels (Fig. 6B, filled triangle). Application of 100 μm Zn²⁺ on cells expressing Ca_V3.3 channels resulted in a decrease in the amplitude of the first inward Ca²⁺ currents, due to the Zn²⁺ inhibition of Ca_V3.3 channels, associated with a significant slowing of the interspike phase that can be best visualized near the fourth spike (Fig. 6B and inset). As a consequence of the slowed deactivation, the inward Ca²⁺ currents obtained after the fifth spike were larger in the presence of Zn²⁺ than those obtained in control conditions, resulting in a net increase in the total Ca²⁺ current. By contrast, only a sizeable Ca²⁺ current reduction was obtained in the presence of Zn²⁺ on cells expressing Ca_V3.1 and Ca_V3.2 channels (Fig. 6C). To quantify these effects, the integral of the total Ca²⁺ current generated by this voltage-command firing activity was measured (Fig. 6D). In the presence of Zn²⁺, the total Ca²⁺ current was significantly reduced for Ca_V3.1 channels (39 ± 19%, n = 5), for Ca_V3.2 channels (81 ± 9%, n = 4) and strongly increased for Ca_V3.3 channels (62 ± 23%, n = 15). These results suggest that Zn²⁺ may differentially modulate the firing activity of neurons expressing distinct populations of Ca_V3 channels.

Figure 6. Zinc modulation of the Ca_V3.1, Ca_V3.2 and Ca_V3.3 currents during thalamic relay cell-like firing activities.

A, the top trace illustrates the generic firing activity of a thalamo-cortical relay cell, which was used as voltage command to perform action potential (AP) clamp experiments. B, time course of the Ca_V3.3 current in control condition (upper trace) and after 100 μm Zn²⁺ application. The current traces obtained for the fourth AP were superimposed (see inset). C, time course of the Ca_V3.1 and Ca_V3.2 currents in control condition and after Zn²⁺ application. Note that 1 μm Zn²⁺ was used to modulate Ca_V3.2 current. D, bar graphs showing the normalized integral of the Ca²⁺ currents (percentage of control: I_Ca,T percentage) generated during thalamic relay cell-like activities in the presence of 1 μm Zn²⁺ for Ca_V3.2 current, 100 μm Zn²⁺ for Ca_V3.1 and Ca_V3.3 currents.

Properties of Ca_V3.3-related action potentials (APs) in the presence of Zn²⁺

Overexpression of Ca_V3.3 channels in NG 108-15 cells results in spontaneous firing of action potentials (Chevalier et al. 2006). We therefore took advantage of this experimental cellular model to evaluate the modulatory action of Zn²⁺ in current clamp conditions on Ca_V3.3 related APs. Zn²⁺ inhibition of Ca_V3.3 channels in NG 108-15 cells was similar to that described in HEK-293 cells since Ca_V3.3 current amplitude was inhibited by ∼30% in the presence of 100 μm Zn²⁺ and the steady-state activation was shifted by ∼10 mV (V_0.5 = −45 ± 0.7 mV and −55 ± 0.5 mV for control and in the presence of Zn²⁺, respectively, n = 8). Similar to that described in HEK-293 cells, inactivation kinetics of Ca_V3.3 current was significantly slowed down in the presence of 100 μm Zn²⁺ (171 ± 20 ms and 240 ± 24 ms for control and Zn²⁺, respectively, n = 8) and deactivation kinetics, measured at −80 mV, was also largely slowed down (5.3 ± 1.4 ms and 73.7 ± 8.0 ms for control and in the presence of Zn²⁺, respectively, n = 11). In current clamp conditions, application of 10 μm Zn²⁺ (as well as 100 μm Zn²⁺, not shown) significantly increased the duration of stimulated APs, as evidenced by the measurement of the AP duration at −40 mV (Δt₋₄₀ value, Fig. 7A and B). Similarly, this modulatory effect of Zn²⁺ on the Ca_V3.3 channel activity was retrieved on spontaneous APs (Fig. 7C–E). Application of 10 μm, or 100 μm Zn²⁺ (not shown), affected neither the appearance of spontaneous APs (Fig. 7C) nor the resting membrane potential, whereas the AP duration was significantly increased and fully reversible upon wash-out (Fig. 7D and E).

Figure 7. Zinc modulation of Ca_V3.3-induced action potentials (APs) in neuroblastoma NG 108-15 cells.

A, superposition of typical examples of stimulated APs recorded before (regular line), during (bold line) and after (dotted line) application of 10 μm of Zn²⁺. APs were elicited by an injected current (I_c) of 0.5 nA during 4 ms. The change in AP duration was measured at −40 mV (Δt₋₄₀), which corresponds to the plateau potential (Chevalier et al. 2006). B, plot of the change in Δt₋₄₀ with time. Note that the increase in AP duration was concomitant with the Zn²⁺ application and fully reversed after its wash-out. C, example of a long time range recording of spontaneous APs obtained before, during (black bar) and after application of 10 μm of Zn²⁺. D, superposition of spontaneous APs recorded before (regular line), during (bold line) and after (dotted line) application of 10 μm Zn²⁺. E, normalized change in Δt₋₄₀ (Δt₋₄₀/Δt_{−40 Ctrl}) during superfusion of 10 μm of Zn²⁺ (black bar) and after wash-out (hatched bar).

Discussion

In the present study, we describe that Zn²⁺ differentially modulates the three Ca_V3 channel isotypes. Zn²⁺ preferentially inhibits Ca_V3.2 channels with an IC₅₀ in the submicromolar range (∼0.8 μm), which is 100- and 200-fold lower than that for Ca_V3.1 and Ca_V3.3 channels, respectively. Zn²⁺ inhibition of Ca_V3 channels is associated with a negative shift of both steady-state activation and inactivation properties, with the exception of the Ca_V3.2 steady-state activation properties that are unchanged. An important finding of our study is that Zn²⁺ induces a significant slowing of inactivation kinetics of both Ca_V3.1 and Ca_V3.3 currents, as well as a drastic slowing of the deactivation kinetics of Ca_V3.3 current. Consequently, action potential clamp experiments in Ca_V3.3-expressing cells reveal that Zn²⁺ can induce a significant increase in the Ca²⁺ entry, especially during the depolarizing after-potential interval. In neuroblastoma NG 108-15 cells, duration of the action potentials mediated by Ca_V3.3 channels is significantly increased, indicating further that Zn²⁺ acts overall as an opener of Ca_V3.3 channels. This dual modulation of Ca_V3.3 channels by Zn²⁺ may represent a strategy to identify Ca_V3.3 channel activity in native neurons. Altogether, we conclude that the differential modulation of T-type Ca²⁺ channels by Zn²⁺, which is a potent physiological modulator, may significantly impact neuronal excitability.

Zn²⁺ preferentially inhibits Ca_V3.2 channels

Ca_V3.2 channels are blocked by submicromolar concentrations of Zn²⁺ (IC₅₀, 0.8 μm) while Ca_V3.1 and Ca_V3.3 channels are unaffected by such Zn²⁺ concentrations, which indicates that Zn²⁺ is a highly potent and selective blocker of Ca_V3.2 channels. Our data confirm a recent study by Jeong et al. (2003) who reported an IC₅₀ of 2.3 μm for Zn²⁺ on rat Ca_V3.2 channels. As a consequence, Zn²⁺, like nickel (Lee et al. 1999), may be useful to pharmacologically distinguish between Ca_V3.2 channels and the other T-type Ca²⁺ channels (Ca_V3.1 and Ca_V3.3) in native tissues. In addition, low concentrations of Zn²⁺ can also be used to discriminate T-type from high voltage-activated (HVA) Ca²⁺ currents in dorsal root ganglion (Busselberg et al. 1992) and sympathetic pelvic neurons (Jeong et al. 2003) in which T-currents are mostly carried out by the Ca_V3.2 subunit (Bourinet et al. 2005). Many other ionic channels, like Na⁺ channels (Amuzescu et al. 2003), high-voltage activated Ca²⁺ channels (Easaw et al. 1999), K_V1.5 (Zhang et al. 2001), K⁺ channels (K_V1.3: Teisseyre & Mozrzymas, 2002; IK_SO and TASK3 (Clarke et al. 2004) and Cl⁻ channels (Pahapill & Schlichter, 1992) are affected by Zn²⁺ concentrations (10–100 μm) larger than those acting on Ca_V3.2 channels, indicating further that this T-channel subtype represents one of the most Zn²⁺-sensitive population of ionic channels. The subtype specificity of Zn²⁺ inhibition on Ca_V3 channels is reminiscent of that reported for tetrodotoxin-resistant Na⁺ channels since Na_V1.5 (IC₅₀, 9 μm; White et al. 1993) is significantly more sensitive to Zn²⁺ than Na_V1.8 and Na_V1.9 subtypes (IC₅₀, 300 μm; Kuo et al. 2004). Also, Zn²⁺ has differential effects on the various members of the ASIC family (Baron et al. 2002) and of the two-pore domain K⁺ channel family since TASK-3 channels are potently inhibited (IC₅₀, 9 μm; Clarke et al. 2004), while TREK-2 are activated (EC₅₀, 85 μm: Kim et al. 2005). The concentration of Zn²⁺ at synapses of the CA3 region of the hippocampus is estimated to be within the range of 100–300 μm (Frederickson et al. 1983; but see Frederickson et al. 2006). Indeed, such concentrations may exert significant tonic inhibitory effects not only on Ca_V3.2 channels, but also on Ca_V3.1 and Ca_V3.3 channels, as well as interfere with the gating behaviour of Ca_V3.1 and Ca_V3.3 channels.

The electrophysiological analysis of the Zn²⁺ effects on recombinant T-type Ca²⁺ channels revealed isotype-specific modulatory effects, probably involving multiple binding sites. Zn²⁺ may be considered as an allosteric modulator of Ca_V3 channels. Both the alterations in the channel gating and the reduction of the currents are likely to occur under physiological conditions and may contribute to the modulatory effects of Zn²⁺ on neuronal activity. Zn²⁺ induces a leftward shift of the steady-state inactivation curves of each Ca_V3 channel. This voltage-dependent inhibition would be particularly relevant in cells that fire action potentials because more depolarized potentials would enhance the potency of blockade. In addition, we found that T-channel inhibition by Zn²⁺ is not use-dependent, suggesting that Zn²⁺ does not necessarily interfere with T-channels in the open state. We therefore suggest that Zn²⁺ preferentially binds to resting Ca_V3 channels.

Zn²⁺ potentiates Ca_V3.3 channels

Another important finding of this study is that Zn²⁺ modifies the gating properties of Ca_V3 channels, especially Ca_V3.3 channels. The reduction of the current amplitude is accompanied by significant Ca_V3 channel kinetic changes upon Zn²⁺ treatment, especially faster activation kinetics (Ca_V3.1), and slowing of inactivation kinetics (Ca_V3.1 and Ca_V3.3) and deactivation kinetics (Ca_V3.3). At first glance, Zn²⁺ would be presented as a Ca_V3.3-channel blocker (see also Jeong et al. 2003). However, our study reveals that Zn²⁺ acts as a mixed blocker/opener of Ca_V3.3 channels. Slowing of Ca_V3.3 deactivation may be caused by zinc binding to a site of the channel pore that does not exclusively block ion flux, but rather prevents activation gate closure upon repolarization. In good agreement with this hypothesis, Kang et al. (2006) identified an extracellular binding site for Ni²⁺ ions on Ca_V3.2 channels, within the S3–S4 linker of domain I, that is critical for Ni²⁺ block and channel gating. Such a site for zinc binding may account for allosteric modulation of Ca_V3 channel gating, especially Ca_V3.3. Alternatively, Talavera & Nilius (2006) demonstrated that mutations in the S6 segments (e.g. M1532I in Ca_V3.1) can affect the rates of both deactivation and inactivation. Binding of zinc to such a site in the channel pore may also possibly account for the various zinc effects on kinetics and steady-state properties. The slowing of both inactivation and deactivation kinetics of Ca_V3.3 current in the presence of Zn²⁺ contributes to a net increase in the Ca²⁺ current, as identified in action potential clamp experiments. Furthermore, current-clamp experiments performed in NG 108-15 cells that generate spontaneous APs directly related to Ca_V3.3 channel activity (Chevalier et al. 2006) reveals a significant broadening of the Ca_V3.3-related APs. These data confirm the influence of deactivation kinetics in setting up AP properties (Chemin et al. 2002). This Zn²⁺ modulation is likely to occur in cells expressing native Ca_V3.3-channels, such as reticular thalamic (nRT) neurons (Talley et al. 1999; Joksovic et al. 2005). It is therefore tempting to suggest that Zn²⁺-modulated Ca_V3.3 channels may contribute to increased Ca²⁺ entry and excitability in nRT neurons. Whether Zn²⁺ could be used as a diagnostic tool to identify functional Ca_V3.3 channels in native neurons is an attractive hypothesis to be tested.

In conclusion, we have demonstrated that Zn²⁺ differentially modulates the three Ca_V3 channels: it is a preferential blocker of Ca_V3.2 channels and it alters the gating behaviour of Ca_V3.3 channels. It is well known that Zn²⁺ is present in many regions of the nervous system, such as mossy fibre terminals of the hippocampus (Frederickson et al. 1983) where the various T-type Ca²⁺ channels are also present. The modulatory effects on T-channels occurs at Zn²⁺ concentrations that are of physiological relevance in the CNS and are likely to have an impact on neuron excitability. Any variation of Zn²⁺ concentration during neurotransmitter release may significantly modulate the electrical behaviour of the various T-channel expressing neurons. Alteration of Zn²⁺ homeostasis also occurs in the brain during various disease states. For instance, reduction in Zn²⁺ concentration is linked with the aetiology of epileptic seizures (Takeda, 2001; Mathie et al. 2006), which may partly rely on a loss of tonic inhibition of Ca_V3.2 channels. Conversely, increased Zn²⁺ release during neuronal ischaemia (Frederickson et al. 2005) may exert neuroprotective effects through Ca_V3 channel inhibition, as suggested by a recent study (Nikonenko et al. 2005). Overall, it is tempting to suggest that Zn²⁺ modulation of Ca_V3 channels contributes to various physiological and disease states and needs further attention.