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T-type Ca_v3.3 calcium channels produce spontaneous low-threshold action potentials and intracellular calcium oscillations

Marc Chevalier

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Philippe Lory,

Philippe Lory

Institut de Génomique Fonctionnelle, Département de Physiologie, CNRS-UMR 5203, INSERM U661, Université Montpellier I & II, 141, rue de la Cardonille, 34094 Montpellier cedex 5, France

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Chantal Mironneau,

Chantal Mironneau

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Nathalie Macrez,

Nathalie Macrez

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Jean-François Quignard,

Jean-François Quignard

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Marc Chevalier,

Marc Chevalier

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Philippe Lory,

Philippe Lory

Institut de Génomique Fonctionnelle, Département de Physiologie, CNRS-UMR 5203, INSERM U661, Université Montpellier I & II, 141, rue de la Cardonille, 34094 Montpellier cedex 5, France

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Chantal Mironneau,

Chantal Mironneau

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Nathalie Macrez,

Nathalie Macrez

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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Jean-François Quignard,

Jean-François Quignard

Laboratoire de Signalization et Interactions cellulaires, CNRS UMR 5017, Université Bordeaux II, UFR Sciences Pharmaceutiques, 146 rue Léo Saignat, 33076 Bordeaux cedex, France

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First published: 16 May 2006

https://doi.org/10.1111/j.1460-9568.2006.04761.x

Citations: 41

Dr J.-F. Quignard, as above.
E-mail: [email protected]

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Abstract

The precise contribution of T-type Ca²⁺ channels in generating action potentials (APs), burst firing and intracellular Ca²⁺ signals needs further elucidation. Here, we show that Ca_v3.3 channels can trigger repetitive APs, generating spontaneous membrane potential oscillations (MPOs), and a concomitant increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) when overexpressed in NG108-15 cells. MPOs were dependent on Ca_v3.3 channel activity given that they were recorded from a potential range of −55 to −70 mV, blocked by nickel and mibefradil, as well as by low external Ca²⁺ concentration. APs of distinct duration were recorded: short APs (sAP) or prolonged APs (pAP) with a plateau potential near −40 mV. The voltage-dependent properties of the Ca_v3.3 channels constrained the AP duration and the plateau potential was supported by sustained calcium current through Ca_v3.3 channels. The sustained current amplitude decreased when the resting holding potential was depolarized, thereby inducing a switch of AP shape from pAP to sAP. Duration of the [Ca²⁺]_i oscillations was also closely related to the shape of APs. The Ca_v3.3 window current was the oscillation trigger as shown by shifting the Ca_v3.3 window current potential range as a result of increasing the external Ca²⁺ concentration, which resulted in a corresponding shift of the AP threshold. Overall, the data demonstrate that the Ca_v3.3 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations. The functional expression of Ca_v3.3 channels can generate spontaneous low-threshold calcium APs through its window current, indicating that Ca_v3.3 channels can play a primary role in pacemaker activity.

Introduction

Calcium influx through voltage-gated calcium channels actively participates in cellular depolarization and simultaneously acts as an intracellular messenger. Calcium channels are involved in a variety of processes from cellular excitability to gene or protein regulation. Although high-voltage-gated calcium channels have been widely studied, little is known about the role of low-voltage- T-type calcium channels in the control of the membrane potential and intracellular Ca²⁺ signalling that they generate (Perez-Reyes, 2003). Studies have suggested that T-type Ca²⁺ channels induce action potential (AP) burst firing activity and low-threshold Ca²⁺ spikes triggered by hyperpolarization-activated cationic channels, I_h (Wilcox et al., 1988; Bal & McCormick, 1993; Huguenard, 1996). T-type Ca²⁺ channels are encoded by three genes (Ca_v3.1, Ca_v3.2 and Ca_v3.3; for reviews see Lacinova et al., 2000; Perez-Reyes, 2003). Each Ca_v3 isotype has distinct electrophysiological characteristics and, among them, Ca_v3.3 channels display unique features. Its current displays slow activation and inactivation kinetics that generate a sustained Ca²⁺ current under a prolonged cellular depolarization (Monteil et al., 2000; Frazier et al., 2001). Ca_v3.3 channels also produce a large window current in the membrane potential's range of −60 to −40 mV, in which a fraction of the channels are always open and do not inactivate (Lee et al., 1999). This property would enable Ca_v3.3 channels to induce a sustained increase in the intracellular Ca²⁺ concentration [Ca²⁺]_i, as reported for skeletal muscle Ca_v3.2 channels (Bijlenga et al., 2000). Ca_v3.3 window current could contribute to depolarization of the cell membrane and to regulation of neuron excitability (Hughes et al., 1999; Pignatelli et al., 2005). Although the electrophysiological properties of Ca_v3.3 channels suggest that they have a prominent role in cellular excitability (Chemin et al., 2002a), it is unknown whether Ca_v3.3 channels can be a primary trigger of burst firing activities. Indeed, in native cells expressing Ca_v3.3 channels, their intrinsic role in cellular excitability and Ca²⁺ homeostasis is occluded by several other ionic conductances that also contribute to neuronal firing activity (Crunelli et al., 2005).

In this study, we have investigated the ability of Ca_v3.3 channels intrinsically to initiate AP firing and [Ca²⁺]_i oscillations in NG108-15 neuroblastoma cells transfected with a cDNA coding for human Ca_v3.3 channels. Our hypothesis was that a Ca_v3.3 window current would be capable of triggering spontaneous membrane potential oscillations (MPOs) and possibly APs. We report that spontaneous MPOs and [Ca²⁺]_i oscillations were correlated with the functional expression of Ca_v3.3 channels and occurred independently of any other depolarizing channel activity. The MPO properties, AP waveforms and [Ca²⁺]_i oscillations are shown to be dependent on the electrophysiological properties of Ca_v3.3 channels. Together, the results show that functional expression of Ca_v3.3 channels may produce pacemaker activity and [Ca²⁺]_i oscillations.

Materials and methods

Cell preparation

The clonal NG108-15 cell line, originally formed by Sendai virus-induced fusion of the mouse neuroblastoma clone N18TGG-2 and the rat glioma clone C6 BV-1, was a generous gift of IdRS (Suresnes, France). Cells were grown in plastic dishes with Dubelcco's modified Eagle's medium supplemented with 7.5% fetal bovine serum (v/v), 2 mm glutamine, hypoxantine-aminopterin-thymidine complement and 10 000 U/mL penicillin/streptomycin in an humidified environment of 5% CO₂, 95% air at 37 °C. At confluence, cells were mechanically resuspended, diluted three-fold and plated in a new plastic dish. For patch-clamp experiments, cells were seeded at 60 000 cells/mL on a glass slide and used within the next 3 days.

Transfection

Four hours after seeding at 60 000 cells/mL, NG108-15 cells were transiently cotransfected with pBK-CMV plasmid constructs that encode for Ca_v3.3a subunit (AF211189, Chemin et al., 2001) and pIRES plasmid constructs that encode for reporter gene CD8 in a ratio of 5 : 1 with JET-PEI reagent according to the manufacturer's recommendations. Twenty-four hours after transfection, positive transfected cells where identified with anti-CD8 antibody-coated beads and further analysed by using voltage-clamp experiments.

Membrane current recording

Voltage-clamp and membrane current recordings were made with a standard patch-clamp technique using a List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). Whole-cell recordings were performed with patch pipettes having resistances of 2–5 MΩ. Membrane potential and current records were stored and analysed on a PC (Pclamp system, Axon Instruments, Foster City, CA, USA). Junction potential was electronically compensated for before acquisition with EPC-7 patch-clamp amplifier (7.1 ± 1 mV, n = 5, for K solution and 4.5 ± 0.8 mV, n = 4 for Cs solution). Cell capacitance was determined in each cell tested by imposing 10-mV hyperpolarizing steps from the holding potential (−70 mV) and analysing the amplitude and the time course of the recorded currents. Current density was expressed as the maximum amplitude of the current per capacitance unit (pA/pF). The currents were measured by a test pulse from −70 mV to 0 mV, a representative value for peak AP. The kinetics of inactivation of the current was calculated with a mono-exponential function [f(t) = A exp(–t/τ) + C) according to the Pclamp set-up. Steady-state inactivation experiments were performed by a double pulse protocol. The holding potential was −70 mV, and a test pulse to 0 mV was preceded by a prepulse of 5-s duration and of variable amplitude (from −100 mV to 10 mV). A steady-state activation curve was constructed from the current–voltage relationship, which was fitted using a combined Boltzmann and linear Ohmic relationship as described by Chemin et al., 2002a, b). Activation and inactivation curves were fitted with a Boltzmann equation, which gives the potential for half-activation and half-inactivation. Recovery from inactivation experiments was performed by a double pulse protocol. The holding potential was −70 mV, and a test pulse to 0 mV was preceded by a prepulse of 500 ms at 0 mV. The interpulse interval was of variable duration (0–3 s).

Cells were superfused with an extracellular pseudo-physiological solution (Ca solution) containing (mm): 130 NaCl, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 11 glucose, 10 HEPES and 0.2 µm tetrodotoxine (TTX), adjusted to pH 7.4 with NaOH. In some experiments, CaCl₂ was omitted, increased to 8 mm or substituted by BaCl₂ or SrCl_2. The basic pipette solution (K solution) contained (mm): 5 NaCl, 50 KCl, 65 K₂SO₄, 2 MgCl₂, 2 ATP and 10 HEPES, adjusted to pH 7.3 with KOH. To record T-type current, without contamination from the potassium channel, the following intracellular solution (Cs solution) was used: (mm): 130 CsCl, 10 EGTA, 5 ATPNa₂, 2 MgCl₂ 10 HEPES, adjusted to pH 7.3 with CsOH. Drugs were applied on the cells via pressure ejection from a glass pipette. All experiments were performed at 32 ± 2 °C.

Intracellular calcium concentration measurement

Measurements of [Ca²⁺]_i were performed with an indo-1 set-up as described by Quignard et al. (2001). Briefly, cells were preloaded with the membrane-permeable indo-1 AM (1 µm) for 30 min; 50 µm indo-1 was added to the pipette solution and entered the cells after establishment of the whole-cell recording mode. [Ca²⁺]_i was estimated from the 405/480-nm fluorescence ratio using a calibration determined within cells.

Chemicals

Antibodies raised against CD8 were purchased from Dynal (Compiegne, France). Mibefradil was a generous gift of Roche (Basel, Switzerland). All other chemicals were purchased from Sigma (St Louis, MO, USA).

Statistics

Values are given as means ± SEM. A Student's t-test was performed to estimate the significance of the differences between two mean values. A value of P < 0.05 was considered significant.

Results

Ca_v3.3 channel activity induces MPOs

Figure 1 shows that in the current-clamp configuration (K/Ca solutions), NG108-15 cells expressing Ca_v3.3 channels produced MPOs. In ∼50% cells, an artificial current was injected to hyperpolarize the membrane potential below −55 mV in order to allow de-inactivation of T-type current and to trigger MPOs. These MPOs were considered to be of low threshold as they were recorded from −70 to −55 mV. They exhibited different waveforms that we have classified in three classes according to the shape and the length of the so-called ‘action potentials’. Short APs (sAP, Fig. 1A) displayed a rather fast repolarization phase shorter than 300 ms. Prolonged APs (pAP, Fig. 1B) presented a delayed repolarization with an inflexion point leading to a short plateau phase (duration of less than 1500 ms). Long pAP (Fig. 1C) showed a long plateau phase of several seconds around −40 mV. These different types of oscillations were also recorded in the cell-attached mode (data not shown, n = 15).

Details are in the caption following the image — **Figure 1**
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Spontaneous membrane potential oscillations in Ca_v3.3-transfected NG108-15 cells. (A–C) Typical membrane potential oscillations in transfected NG108-15 cells (current-clamp, records obtained in different cells). Note the different shape of spontaneous activity (A) sAP, (B) pAP and (C) long pAP. Hatched lines indicate the level of potential noted on the left. Measurements were performed in Ca/K solution.

Several experimental findings indicated further that these MPOs could be attributed to the activity of Ca_v3.3 T-type Ca²⁺ channels. Indeed, such MPOs were never observed on nontransfected cells, at any potential tested (from −30 to −90 mV, n = 29, Fig. 2A). Only transfected cells displaying a T-type current density higher than ∼ 11 pA/pF (75% of transfected cells) showed MPOs (Fig. 2B). Indeed, mean current density of oscillating cells was around 20 pA/pF, whereas nonoscillating cells had a current density of about 7.5 pA/pF. Furthermore, MPOs were inhibited by T-type Ca²⁺ channel inhibitors. Nickel at 300 µm, a concentration that inhibits > 50% of the Ca_v3.3 T-type current, occluded oscillations (Fig. 2C) whereas 30 µm nickel, a concentration which weakly inhibits Ca_v3.3 current (data not shown; see also Perez-Reyes, 2003), did not alter the oscillations (Fig. 2D). Oscillations were also inhibited by mibefradil (1 µm) and pimozide (1 µm) and by lowering the extracellular Ca²⁺ concentration (0.1 mm) (n = 3 for each condition, data not shown). An involvement of other conductances in generation of MPOs was unlikely as they were inhibited neither by a (1) Na⁺ channel inhibitor, TTX (1 µm); (2) nor by an L-type Ca²⁺ channel inhibitor, oxodipine (1 µm, data not shown, n = 3); (3) nor by substitution of Cl^– and Na⁺ ions by the nonpermeable agents methane sulphonate or NMDG, respectively, (4) nor by the nonselective cationic channel inhibitor flufenamic acid (1 µm) (data not shown, n = 3). Additional experiments showed that MPOs could also be recorded in the presence of barium as charge carrier, and in the presence of extracellular CsCl (10 mm), which is known to block I_h (Pape, 1996) (Fig. 2E). In all cases, sAP and pAP were recorded. The Ca_v3.3-induced MPOs were not dependent on cell type, given that HEK-293 cells transfected with the Ca_v3.3 cDNA also showed sAP or pAP oscillations with similar properties (n = 4).

Figure 3 shows that [Ca²⁺]_i oscillations were associated with the Ca_v3.3-dependent MPOs. The waveform of [Ca²⁺]_i oscillations was controlled by the shape of the APs. As illustrated in Fig. 3A, long pAP induced concomitant and maintained [Ca²⁺]_i increases of 130 ± 20 nm over the basal value (115 ± 10 nm, n = 20), whereas more rapid APs induced Ca²⁺ oscillations (293 ± 40 nm, n = 10) with peaks of shorter duration, generating a maintained rise of the basal [Ca²⁺]_i (Fig. 3B). Similar to that obtained for the MPOs, the [Ca²⁺]_i oscillations were inhibited by lowering extracellular Ca²⁺ concentration as well as by reducing the membrane potential beyond −70 mV (Fig. 3B). These Ca²⁺ oscillations were also inhibited by nickel and mibefradil, further supporting that Ca_v3.3 T-type channels were the pathway of Ca²⁺ entry. Together, these data indicated that Ca_v3.3 channel expression may be responsible for significant changes in both membrane potential and [Ca²⁺]_i.

MPOs are related to the electrophysiological properties of the Ca_v3.3 channel

As the different types of MPOs could be observed in the same cell, we hypothesized that the membrane potential could influence the MPO waveforms. In a given Ca_v3.3-transfected NG108-15 cell, variations in membrane potential switched the MPO waveform from sAP to pAP and long pAP, as shown in Fig. 4A. Variations of a few millivolts in the membrane potential could considerably modify the MPO waveforms (Fig. 4A). The sAP, pAP and long pAP could be defined by the average membrane potentials at which they were recorded (Fig. 4B), by their rate of firing (Fig. 4C) and by their amplitude (48.6 ± 1.3 mV, n = 145; 57.6 ± 1.5 mV, n = 160; 76.1 ± 1.9 mV, n = 101; for sAP, pAP and long pAP, respectively). Consequently, the frequency of APs decreased whereas their amplitude increased, when hyperpolarizing the cell and shifting AP waveforms from sAP to pAP. Nevertheless, the rising kinetics of the three AP types were similar (25.5 ± 0.5 ms, n = 406). The Ca²⁺ currents, supporting MPOs, were then characterized in a Cs/Ca solution. A step depolarization from −70 to 0 mV revealed typical characteristics to that of the Ca_v3.3 T-type current described earlier (Chemin et al., 2001): kinetics of inactivation, 67.25 ± 9.01 ms (n = 10); potential for half-inactivation (V_0.5 inact), −59.96 ± 0.20 mV (n = 5); half-life recovery from inactivation, 125.10 ± 7.29 ms (n = 4).

To study the properties of the Ca_v3.3 current during APs, we then performed AP clamp experiments using digitized (sAP, pAP) and mock APs that mimicked pAP as waveform stimuli, in Cs/Ca solution. As shown in Fig. 5A, sAP induced only a transient inward current (Fig. 5A, a3) and no such current in nontransfected cells (Fig. 5A, a2). By contrast, a pAP or mock APs with a plateau phase at −40 mV induced a two-component inward current: a transient inward current followed by a sustained inward current that occurs during the plateau phase (Fig. 5A, b3 and c3). By contrast, no such inward current could be observed in nontransfected cells but rather a small residual outward current (Fig. 5A, b2 and c2). The transient inward current was carried out by T-type channels, as it was inhibited by nickel (500 µm, n = 4). The amplitude of the sustained current was dependent on the plateau potential value and the maximum current amplitude was obtained for a plateau potential of between −50 and −30 mV, which correspond to the value of the plateau potential of pAP. Indeed, this current could not be recorded at −70 mV (where only a rapid deactivating current was recorded, Fig. 5A, c3). Moreover, the amplitude of the sustained current depended on the resting membrane potential. Detectable at a holding potential of −70 mV, the sustained current was inactivated at a holding potential of −50 mV (Fig. 5B and C). The lack of plateau potential displayed by sAP could be explained by the depolarized resting membrane potentials from which they are triggered and at which the sustained current was inhibited. In the next set of experiments, the sustained Ca_v3.3 current was characterized further. It was of similar amplitude after substitution of Ca²⁺ by Ba²⁺ or Sr²⁺ ions, and it was significantly enhanced when the extracellular Ca²⁺ or Ba²⁺ concentration was increased to 10 mm. It was inhibited by mibefradil (data not shown) and nickel (Fig. 5D) in a dose-dependent manner. The effect of Cl^– or Na⁺ ions was not tested as the sustained current was not significantly altered after substitution of these ions by the nonpermeable agents methane sulphonate or NMDG, respectively. It was not a Ca²⁺-activated current, as it could be recorded after substitution of Ca²⁺ by Ba²⁺ (Fig. 5D) or chelation of intracellular Ca²⁺ by EGTA (10 mm) (data not shown). Furthermore, it was not inhibited by the nonselective cationic channel inhibitor flufenamic acid (1 µm). Additional experiments confirmed that this sustained Ca²⁺ current was specific for the Ca_v3.3 channels given that nontransfected cells or cells transfected with Ca_v3.1 channels did not display any sustained inward current in this experimental situation (n = 5, data not shown). Overall, these results indicated that the pAP plateau was supported by Ca_v3.3 T-type Ca²⁺ channels.

Role of Ca_v3.3 window current in triggering MPOs

We measured [Ca²⁺]_i and steady-state Ca²⁺ entry using K/Ca solution, in transfected NG108-15 cells loaded with indo-1. A 100-ms step depolarization induced a large inward T-type current concomitant with a large [Ca²⁺]_i increase (195 ± 17 nm, n = 12, from a basal level of 120 ± 12 nm, Fig. 6A). The variation in [Ca²⁺]_i amplitude was voltage-dependent and the voltage/[Ca²⁺]_i relationship was similar to the voltage/current relationship (Fig. 6B). Analysis of the steady-state activation and inactivation properties of the Ca_v3.3 T-type Ca²⁺ channels in NG108-15 cells revealed that a steady-state Ca²⁺ current, referred to as a ‘window current’, could exist in the membrane potential range where the MPOs were elicited (Fig. 6C). Such a T-type Ca²⁺ channel window current could control the resting membrane potential as it corresponds to a continuous depolarizing current (Crunelli et al., 2005). Such an effect on [Ca²⁺]_i was assessed by using a ramp potential from −85 to 0 mV in voltage-clamp conditions at a slow depolarization speed (0.5 mV/s; Fig. 6D). In Ca_v3.3-transfected NG108-15 cells, this ramp protocol induced a significant increase in the [Ca²⁺]_i of 104 ± 25 nm over the basal level (118 ± 15, n = 6, Fig. 6D). This bell-shaped Ca²⁺ response exhibited large [Ca²⁺]_i increase in the potential range of the window current. A [Ca²⁺]_i increase of similar characteristics was also observed when the ramp protocol was reversed (from 0 to −85 mV; hyperpolarization speed, 0.5 mV/s; [Ca²⁺]_i increase, 98 ± 12, n = 5, Fig. 6E). The [Ca²⁺]_i increase was inhibited by nickel (500 µm, 20 nm ± 10 nm, n = 3) and absent in nontransfected cells (6.1 ± 1 nm, n = 6) stimulated with a depolarizing or hyperpolarizing ramp. These results suggested that the window current mediated a [Ca²⁺]_i increase between −70 and −40 mV that could explain the triggering of MPOs.

To explore further the relationships between the Ca_v3.3 window current and the MPOs, the voltage range of the Ca_v3.3 window current was shifted by modifying the external Ca²⁺ concentration. Increasing extracellular Ca²⁺ concentration from 2 mm to 8 mm induced a significant +9.1 ± 1.9 mV (n = 6) shift of the potential of half-activation (V_0.5 act) (Fig. 7A and B) and consequently of the window current potential range. The threshold potential required to obtain similar spontaneous oscillations as at 8 mm compared with 2 mm (see Fig. 7C) was shifted significantly toward depolarized potentials by +8.1 ± 2.4 mV (n = 5). These data are in good agreement with a role of the Ca_v3.3 window current in controlling MPOs. Similar results were obtained in the presence of Ba²⁺ as charge carrier when the Ba²⁺ concentration was increased from 2 to 10 mm (data not shown). Overall, these data indicate that the spontaneous MPOs described are directly triggered by the activity of Ca_v3.3 T-type Ca²⁺ channels and that the MPO waveform reflects the specific electrophysiological properties of the Ca_v3.3 T-type Ca²⁺ channels, including those of its window current.

Discussion

The main finding of this study is that Ca_v3.3 channels can generate spontaneous and repetitive low-threshold Ca²⁺ APs of various shape associated with intracellular Ca²⁺ oscillations. These membrane potential and [Ca²⁺]_i oscillations are closely related to the channel's electrophysiological properties and do not involve other pacemaker currents. The Ca_v3.3 window current is sufficient to provide the depolarizing drive triggering APs during each cycle of firing.

Functional expression of the Ca_v3.3 channels in proliferative NG108-15 neuroblastoma cells results in a large T-type Ca²⁺ current, as reported earlier (Chemin et al., 2001), without contamination by high-threshold Ca²⁺ currents (Lukyanetz, 1998). This Ca_v3.3 current displays slow inactivation kinetics and a large window current component in the range of the resting membrane potential as in HEK-293 cells (Lee et al., 1999; Monteil et al., 2000). We document here that expression of Ca_v3.3 channels induces spontaneous low-threshold MPOs. MPOs result from the activation of T-type channels because (1) they are present only when an inward Ca²⁺ current is present, (2) these oscillations become apparent in the hyperpolarized potential range where Ca_v3.3 channels are not inactivated (i.e. below −50 mV) and (3) they are inhibited by nickel and mibefradil. Furthermore, MPOs can be recorded on intact cells in the cell-attached mode (Fenwick et al., 1982). In nontransfected NG108-15 cells, a small endogenous T-type current is recorded (Lukyanetz, 1998; Chemin et al., 2002a, b). Ca_v3.2 channels support this current because it inactivates rapidly and it is blocked by low nickel concentration (30 µm, Chemin et al., 2002a, b; and our unpublished observations). We show that this endogenous T-type current does not contribute to oscillation as 30 µm nickel is unable to inhibit oscillations. Moreover, oscillations are not inhibited by Na⁺ and high threshold-activated Ca²⁺ channel blockers (TTX, oxodipine) and the shape, frequency and threshold of these MPOs are significantly different from those reported for the firing activity induced by high-threshold-activated Ca²⁺ channels (Takahashi et al., 1999).

In several cell types, low-threshold MPOs are associated with I_h activity (Pape, 1996). Other studies, however, have shown that I_h cannot promote oscillations unless a depolarizing current is present between APs (Feigenspan et al., 1998). In NG108-15 cells, no functional I_h current could be recorded and the application of extracellular CsCl, an inhibitor of I_h, did not inhibit oscillations. ZD7288, a well-known inhibitor of I_h, could not be used as it is also described as an inhibitor of T-type channels (Felix et al., 2003). We then provide several arguments supporting that, in transfected cells, pacemaker activity relies on the T-type window current. Sodium or chloride ions are not involved in pacemaker activities as their substitution by NMDG and methane sulphonate, respectively, does not impede the oscillations. The potential range of spontaneous APs is similar to that of the T-type window current. When the window current potential range is shifted to more depolarized values, the potential between APs is similarly shifted (for both sAP and pAP). These data strongly indicate that the depolarizing current between APs is carried out by Ca²⁺ through Ca_v3.3 channels. More importantly, we were able to visualize the steady-state Ca²⁺ entry through the T-type window current by direct [Ca²⁺]_i measurements. Using slow ramp protocols in voltage-clamp experiments, a significant [Ca²⁺]_i increase occurs between −70 and −40 mV, which corresponds to the potential range of the T-type window current. Nonvoltage-gated conductance could not account for the shape of this signal because in such a case the [Ca²⁺]_i increase at −80 mV should be more important than at −50 mV due to the Ca²⁺ driving force. Nevertheless, the small voltage range difference between Ca²⁺ and the window current signal could be due to accumulation of intracellular Ca²⁺ and the slow kinetics of Ca²⁺ extrusion. MPOs are not specific to NG108-15 cells; they can also be recorded in transfected HEK-293 cells, which lack endogenous pacemaker currents (Ludwig et al., 1999). Together, these results demonstrate that Ca_v3.3 T-type Ca²⁺ channels can play a primary role in pacemaker activity. These observations comply well with a mathematical model, which further indicates that MPOs are present only if there is an appropriate ratio of Ca_v3 current to leak current (Williams et al., 1997; Hughes et al., 1999). A small window current may not be able to overcome background hyperpolarizing currents and to induce oscillations. Indeed, the MPOs were recorded only in cells that showed large T-current density. Together, our results demonstrate that the activity of Ca_v3.3 channels is important in inducing and maintaining oscillations. The ability of T-type current to induce MPOs could be enhanced when there is an increase in its density as a result of their specific subcellular localization as in dendrites of reticularis thalami neurons (Joksovic et al., 2005).

The shape of APs within MPOs correlates well with the electrophysiological properties of Ca_v3.3 channels. The activation kinetics of the Ca_v3.3 current (around 7 ms, Perez-Reyes, 2003) can easily support the rising phase of the APs (which last up to 25 ms). Each AP peak correlates well with Ca_v3.3 peak current, as demonstrated in AP clamp experiments (Fig. 5). The transient repolarization phase of pAP, before the plateau phase, could be primary due to voltage-dependent inactivation of Ca_v3.3 current. Indeed, inactivation kinetics is strongly voltage-dependent and the more the cell is depolarized, the faster is the inactivation of the current. Concomitant activation of outward current(s) could also possibly contribute to this transient repolarization phase. The amplitude of the sAP is smaller than the amplitude of the pAP, in agreement with the lower number of available channels at depolarized potential (as observed on the steady-state inactivation curve). The plateau phase of pAP involves a sustained inward current through Ca_v3.3 channels. The sustained current was recorded only in hyperpolarized cells (below −62 mV). At a more depolarized holding potential, sustained Ca_v3.3 current was drastically reduced. These data provide a likely explanation for the lack of a plateau in more depolarized cells. The nature of the sustained Ca_v3.3 current is rather complex, involving deactivation, inactivation and activation properties (Frazier et al., 2001). At the plateau potential (−40 mV), the kinetics of channel closing is faster than inactivation kinetics, which enables Ca_v3.3 channels to re-open (Frazier et al., 2001). In addition, participation of the Ca_v3.3 window current should be considered at this potential. Finally, although it is known that in subthalamic nucleus or thalamic neurons Ca²⁺-activated currents contribute to the plateau phase (Beurrier et al., 1999; Hughes et al., 2002), we report that in our experimental conditions the plateau phase of pAP does not involve activation of other depolarizing conductances such as sodium-, chloride- or Ca²⁺-activated currents. Furthermore, pAP could also be recorded in HEK-293 cells, indicating further that endogenous background channels are poorly involved in the plateau phase of cells or cellular domains expressing large Ca_v3.3 current density.

We document here that Ca_v3.3 channels participate in the control of [Ca²⁺]_i and Ca²⁺ signalling at different levels. Ca_v3.3 channels can mediate a voltage-dependent increase of [Ca²⁺]_i, as well as a maintained [Ca²⁺]_i increase, in relation to the Ca_v3.3 window current. These data confirm that window current controls not only membrane potential (Crunelli et al., 2005; this study) but also [Ca²⁺]_i. This Ca_v3.3-mediated [Ca²⁺]_i increase is likely to activate various Ca²⁺-dependent mechanisms, as shown for the Ca_v3.2 window current-dependent [Ca²⁺]_i increase in fusion of human myoblasts (Bijlenga et al., 2000). The [Ca²⁺]_i increase-related window current observed in NG108-15 cells (∼100 nm) is greater than that reported for myoblasts, possibly due to the larger amplitude of the Ca_v3.3 window current (Lee et al., 1999). During MPOs, Ca_v3.3 channel induces a maintained increase of [Ca²⁺]_i that is larger than the Ca²⁺ increase induced by a single depolarization. The maintained increase in [Ca²⁺]_i may result from the summation of sequential activation of Ca_v3.3 channels. Overall, these data indicate that Ca_v3.3 channels can generate a variety of Ca²⁺ signals that may differentially impact neuron physiology.

The physiological relevance of these results may be of importance. Ca_v3.3 channels are mainly expressed in the central nervous system, such as in lateral habenula, reticular and latero-dorsal thalamic nuclei. In these nuclei, slowly inactivating T-type currents have been recorded (Wilcox et al., 1988; Bal & McCormick, 1993) that could further be associated with Ca_v3.3 channels (Perez-Reyes, 2003). In those neurons, oscillatory activity has been linked to T-type channel de-activation (for a review see Huguenard, 1996). Similar to our data, the frequency of rebound oscillation was shown to depend on membrane potential, and the duration of each AP was inversely related to its amplitude (Wilcox et al., 1988). In reticular thalamic nuclei, a slow inactivating T-type current, likely to be related to Ca_v3.3 channel, is essential for generation of burst firing activity (Joksovic et al., 2005), confirming that Ca_v3.3 channels can act as a pacemaker current. The spike bursts are prolonged and less self-limiting in these neurons than in neurons that express other types of T-type channels (Mulle et al., 1986; Huguenard & Prince, 1992; Huguenard et al., 1993). Our description of the Ca_v3.3 sustained current may explain the long duration of the burst.

Together, our data demonstrate that Ca_v3.3 channels can generate a pacemaker current triggering low-threshold Ca²⁺ APs and that Ca_v3.3 sustained current regulates intrinsic electrical and [Ca²⁺]_i oscillations. It is therefore tempting to suggest that in cells that express Ca_v3.3 channels, as in reticular thalamic nuclei neurons, Ca_v3.3 currents can directly contribute to the fine tuning of burst duration, as evidenced in NG108-15 cells over-expressing Ca_v3.3 channels.

Acknowledgements

This work was supported by grants from the Centre National de la Recherche Scientifique, the Centre National des Etudes Spatiales and the Association Française contre les Myopathies, France. We thank N. Biendon for secretarial assistance and J. Mironneau for his support, constructive remarks and corrections of the manuscript.

Abbreviations

APs: action potentials, [Ca²⁺]_i, intracellular Ca²⁺ concentration
MPOs: membrane potential oscillations
TTX: tetrodotoxine
sAP: short APs
pAP: prolonged APs

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Citing Literature

Volume23, Issue9

May 2006

Pages 2321-2329

T-type Ca_v3.3 calcium channels produce spontaneous low-threshold action potentials and intracellular calcium oscillations

Abstract

Introduction