Neurons, muscle cells and many other types of cells use electrical signals to exchange information and coordinate their behavior. Proteins known as calcium channels sit in the membrane that surrounds the cell and can generate electrical signals by allowing calcium ions to cross the membrane and enter the cell during electrical activities. Although calcium ions are needed to generate these electrical signals, and for many other processes in cells, if the levels of calcium ions inside cells become too high they can be harmful and cause disease.

Cells have a “feedback” mechanism that prevents calcium ion levels from becoming too high. This mechanism relies on the calcium ions that are already in the cell being able to close the calcium channels. This feedback mechanism has been extensively studied in two types of calcium channel, but it is not known whether a third group of channels – known as Cav3 channels – are also regulated in this way.

Cav3 channels are important in electrical signaling in neurons and have been linked with epilepsy, chronic pain and various other conditions in humans. Cazade et al. investigated whether calcium ions can regulate the activity of human Cav3 channels. The experiments show that these channels are indeed regulated by calcium ions, but using a distinct mechanism to other types of calcium channels. For the Cav3 channels, calcium ions alter the gating properties of the channels so that they are less easily activated . As a result, fewer Cav3 channels are “available” to provide calcium ions with a route into the cell.

The next steps following on from this work will be to identify the molecular mechanisms underlying this new feedback mechanism. Another challenge will be to find out what role this calcium ion-driven feedback plays in neurological disorders that are linked with altered Cav3 channel activity.

Voltage-gated Ca²⁺ channels (VGCCs) are unique among voltage-gated ion channels because the permeant Ca²⁺ ion also acts as an intracellular second messenger, triggering diverse cellular functions (Berridge et al., 2003). VGCCs are therefore involved in neuronal and cardiac excitability as well as in muscle contraction, neurotransmitter release, hormone secretion and gene expression (Berridge et al., 2003; Mangoni and Nargeot, 2008; Catterall, 2011; Simms and Zamponi, 2014; Zamponi et al., 2015). Consequently the modulation of VGCC activity plays a pivotal role in the regulation of cardiac and brain activities and this modulation is controlled by a variety of processes, including intracellular Ca²⁺ itself, which provides an important Ca²⁺-driven feedback control (Eckert and Chad, 1984; Zühlke et al., 1999; Peterson et al., 1999; Liang et al., 2003; Green et al., 2007; Tsuruta et al., 2009; Oliveria et al., 2012; Hall et al., 2013; Zamponi et al., 2015).

VGCCs comprise three distinct subfamilies classified with respect to their biophysical and pharmacological (type), and molecular (Cav) entities: the L-type / Cav1, the N-, P/Q-, R-type / Cav2 and the T-type / Cav3 channels (Ertel et al., 2000). It was well demonstrated that both Cav1 and Cav2 channels are modulated by intracellular Ca²⁺ (Liang et al., 2003; Dick et al., 2008). For the Cav1 / L-type VGCCs, this Ca²⁺ feedback mechanism has been extensively studied in a wide spectrum of biological contexts and a rise in submembrane Ca²⁺ concentration induces complex effects depending on both the Ca²⁺ concentration and the duration of the Ca²⁺ entry (Eckert and Chad, 1984; Zühlke et al., 1999; Peterson et al., 1999; Liang et al., 2003; Green et al., 2007; Tsuruta et al., 2009; Oliveria et al., 2012; Hall et al., 2013). At the millisecond time scale, the Ca²⁺ entry via L-type channels induces a Ca²⁺-dependent inactivation (CDI) characterized by an acceleration of their inactivation kinetics (Eckert and Chad, 1984; Zühlke et al., 1999; Peterson et al., 1999; Liang et al., 2003; Hall et al., 2013). For several seconds to a few minutes of stimulation, the cumulative Ca²⁺ entry induces a decrease of the L-type current amplitude, which is reversible if stimulation ceases for several minutes (Eckert and Chad, 1984; Oliveria et al., 2007, 2012; Hall et al., 2013). For longer period of stimulation, or activation of the ionotropic NMDA receptors, L-type channels are internalized, potentially degraded in lysosomes or recycled to the plasma membrane depending on the amount and the duration of the Ca²⁺ entry (Green et al., 2007; Tsuruta et al., 2009; Hall et al., 2013). This precise Ca²⁺-dependent regulation of the L-type channel activity has a strong physiological role in avoiding cytotoxicity arising from Ca²⁺ overload (Lee et al., 1999; Berridge et al., 2003; Green et al., 2007; Tsuruta et al., 2009; Hall et al., 2013). Consequently, alteration of the Ca²⁺-dependent regulation of the L-type channels is deleterious and has important pathophysiological consequences as observed in the Timothy syndrome (Splawski et al., 2004; Barrett and Tsien, 2008; Blaich et al., 2012; Limpitikul et al., 2014; Dick et al., 2016).

Contrasting with this well-established Ca²⁺-dependent regulation of the L-type channels, it is presently unknown whether a change in intracellular Ca²⁺ concentration is involved in regulating T-type Ca²⁺ channel activity. The low-voltage-activated, T-type/Cav3 channels are specifically activated by small membrane depolarization below the threshold of classical sodium action potentials, producing a Ca²⁺ entry near the resting membrane potential and low-threshold Ca²⁺ spikes (Huguenard, 1996, 1998; Perez-Reyes, 2003; Zamponi, 2016). Importantly, availability of Cav3 channels is critically regulated by the resting membrane potential to control T-type channel activity. Because of the negative range of their steady-state inactivation (V_0.5 near −70 mV), Cav3 channels are partially inactivated (reduced availability) in the range of the resting membrane potential of most neurons and a membrane hyperpolarization (usually triggered by inhibitory postsynaptic events) is needed to allow their recovery from inactivation (de-inactivation) and their subsequent opening (Huguenard, 1996; Perez-Reyes, 2003; Zamponi, 2016). This behavior is of particular importance in many types of neurons, in which Cav3 channels mediate rebound burst firing, especially in the thalamo-cortical circuit, where Cav3 channels control transition between awake and sleep states (Huguenard, 1996; Perez-Reyes, 2003; Beenhakker and Huguenard, 2009; Zamponi, 2016; Tatsuki et al., 2016). Overall, Cav3 channels are involved in the control of the Ca²⁺ homeostasis (Chemin et al., 2000; Bijlenga et al., 2000; Perez-Reyes, 2003), in Ca²⁺-dependent differentiation of neuronal, muscular and neuroendocrine cells (Bijlenga et al., 2000; Mariot et al., 2002; Chemin et al., 2002b), as well as in Ca²⁺ overload toxicity in ischemia (Nikonenko et al., 2005; Bancila et al., 2011; Gouriou et al., 2013). Importantly, an increase in the Cav3 channel activity has been implicated in several diseases including epilepsy, chronic pain, autism and primary aldosteronism (Beenhakker and Huguenard, 2009; Zamponi, 2016). Although it is evident that a tight control of Cav3 channel activity is necessary to maintain Ca²⁺ homeostasis, there is no evidence yet that Cav3 channels are regulated by intracellular Ca²⁺ ions and/or by Ca²⁺ entry.

In this study, we have designed complementary electrophysiological experiments to explore whether the T-type/Cav3 channels are modulated by intracellular Ca²⁺ concentration. We document a feedback control mechanism that relies on Ca²⁺ entry via activated Cav3 channels or nearby Ca²⁺-permeable receptors. We provide evidence that dynamic changes and localized increase in the intracellular Ca²⁺ concentration at the vicinity Cav3 channels control availability of these channels, which underlies this novel regulation.

This study reveals that T-type / Cav3 channels are dynamically regulated by changes in intracellular Ca²⁺ concentration. This novel regulation involves a Ca²⁺-dependent modulation of Cav3 availability. It was unraveled by demonstrating that a rise in submembrane Ca²⁺ at the vicinity of the Cav3 channels can cause a hyperpolarizing shift in the steady-state inactivation, leading to a strong Cav3 current decrease at physiological resting membrane potentials. This localized increase in intracellular Ca²⁺ can be generated by the Cav3 channel activity itself, especially for Cav3.3 channels, or by other routes of Ca²⁺ entry through the plasma membrane as obtained following activation of various Ca²⁺-permeable ionotropic receptors, all inducing a potent inhibition of Cav3.1, Cav3.2 and Cav3.3 currents. Importantly, all the effects described here were observed in 2 mM Ca²⁺-containing saline, which corresponds to the physiological range of extracellular Ca²⁺ concentration in native tissues (Jones and Keep, 1988), and were lost in the presence of intracellular BAPTA.

The Ca²⁺-dependent modulation revealed here was best evidenced with Cav3.3, which allows the largest Ca²⁺ entry among Cav3 channel family (Klöckner et al., 1999; Kozlov et al., 1999; Chemin et al., 2002a; Perez-Reyes, 2003). The decrease in Cav3.3 current amplitude was well correlated with the increase in the frequency of the TP stimulation. The reduction in Cav3.3 current amplitude was ~50% at 1 Hz frequency of stimulation and reached ~80% during neuronal activities in action potential clamp, suggesting that intracellular Ca²⁺ could be an important physiological regulator of Cav3 current. Importantly, this effect was very dynamic both in its induction (T_0.5~18 s) and in its recovery (T_0.5~20 s). Altogether, our results demonstrate that the Cav3 current decrease is linked to the cumulative Ca²⁺ entry through Cav3 channels. This Ca²⁺-dependent inhibition of Cav3 channels was observed only in the presence of external Ca²⁺ indicating that a voltage-dependent component in the mechanism is unlikely to contribute to the reported effect. Importantly, this Cav3 current inhibition induced by the Ca²⁺ entry is observed using intracellular EGTA but not anymore after the cell dialysis with BAPTA. These data reveal that this Ca²⁺-dependent modulation of Cav3 channels involves a localized increase in submembrane Ca²⁺ at the vicinity of the Cav3 channels without the need for a global Ca²⁺ increase (Marty and Neher, 1985; Roberts, 1993; Deisseroth et al., 1996). Interestingly, in the inside-out patch configuration, the direct application of Ca²⁺-containing solutions up to 100 µM to the internal side of the membrane did not produce the current inhibition, suggesting that this phenomenon requires some additional components preserved in the whole-cell configuration.

A key finding of this study is that the current inhibition is closely linked to the resting membrane potential (Vm; see the schematic representation in Figure 7). On one hand, an increase in submembrane Ca²⁺ promotes a strong negative shift in the steady-state inactivation properties of T-type channels, leading to a more prominent inhibition of the Cav3 current at physiological Vm (~ −65/–85 mV; Figure 7). On the other hand, the Ca²⁺ entry via T-type channels is maximal at negative Vm (~ −100 mV), but the consequence of the shift in the steady-state inactivation is minimized at negative Vm (Figure 7). Consequently, the inhibition of the Cav3 current is maximal at physiological resting membrane potentials after stimulation at negative HP. Using this paradigm our data revealed an important finding: a wide range of modulation (~600%) of both Cav3.3 and Cav3.1 currents, but not of Cav3.2 current which was resistant to this modulation.

The lack of Ca²⁺-dependent modulation of Cav3.2 channels using fast TP stimulation protocols could be explained by the specific electrophysiological properties of this Cav3 isoform. It is expected that the Cav3.2 current would generate little cumulative Ca²⁺entry because of its rapid inactivation kinetics combined with its slow recovery from inactivation (Klöckner et al., 1999; Kozlov et al., 1999; Satin and Cribbs, 2000; Chemin et al., 2002a; Perez-Reyes, 2003). Interestingly, the Met1549Ile gain-of-function mutant of the Cav3.2 channel that displays very slow inactivation and deactivation kinetics (Scholl et al., 2015; Daniil et al., 2016), exhibits a significant Ca²⁺-dependent inhibition and a wide range of modulation (~300%). Collectively, our data indicate that the Ca²⁺-dependent mechanism described here can modulate all three Cav3 isoforms. Overall, the amplitude of the modulation (Cav3.3 > Cav3.1 >> Cav3.2 ~ 0) critically depends on the amount of the Ca²⁺ entry and therefore relies on the biophysical properties of each Cav3 isoform.

The large Ca²⁺ entry generated by the activation of ionotropic receptors induces a strong inhibition (~85%) of all three Cav3 currents, including wild-type Cav3.2. We observed this effect with a variety of Ca²⁺-permeable ionotropic receptors, including the purinergic P2X4, the glutamatergic NMDA, the serotoninergic 5-HT3 and the TRP (TRPV1 and TRPA1) receptors. Notably, no inhibition of the T-type current is observed when these experiments are performed in the absence of extracellular Ca²⁺, demonstrating the critical role played by the Ca²⁺ entry in this mechanism of Cav3 inhibition. It is noteworthy that the tsA-201 cells, which derived from HEK-293 cells, endogenously express another class of purinergic receptors, P2Y, which are Gq-coupled receptors and induce a global increase in intracellular Ca²⁺ in response to ATP (Chemin et al., 2000; Fischer et al., 2005). Interestingly, we observed no change Cav3 current properties in pcDNA3 transfected cells (Figure 6b) indicating that Cav3 currents are not inhibited in P2X untransfected cells following ATP application. These data suggest that activation of P2Y receptors is unable to mediate a Ca²⁺-dependent modulation of Cav3 currents further supporting a membrane-delimited mechanism for the Ca²⁺-dependent modulation of Cav3 channels.

Since the initial discovery of T-type currents, it was admitted that these channels were not regulated by intracellular Ca²⁺ ions or by changes in intracellular Ca²⁺ concentration because they do not present CDI (Carbone and Lux, 1984; Fedulova et al., 1985; Bean, 1985; Bossu et al., 1985; Bossu and Feltz, 1986; Dupont et al., 1986). More recently, these findings were confirmed using cloned T-type channels (Staes et al., 2001; McRory et al., 2001; Perez-Reyes, 2003) but see (Lacinová et al., 2006). Indeed, our data showing that the inactivation kinetics are similar at the beginning of the stimulation obtained in external Ca²⁺ and in external sodium (Figure 2e) also support the lack of CDI for Cav3 channels. Accordingly, the important structural motifs for CDI present on L-type channels, as the Ca²⁺ (EF Hand) and the calmodulin (IQ motif) binding sites are absent on the C-terminus of Cav3 channels (Staes et al., 2001; McRory et al., 2001; Perez-Reyes, 2003). Therefore, CDI was considered in pioneer studies as a hallmark to distinguish between L-type and T-type Ca²⁺ currents (Carbone and Lux, 1984; Fedulova et al., 1985; Bean, 1985; Bossu et al., 1985; Bossu and Feltz, 1986; Dupont et al., 1986). Increasing the intracellular Ca²⁺ concentration [Ca²⁺]_INT from 10⁻¹⁰ to 10⁻⁷ M or even to 10⁻⁶ M was classically used to isolate a ‘pure’ T-type current, which presented no change in its inactivation kinetics, whereas the L-type current disappeared because of the acceleration of its ‘run-down’ (Bossu et al., 1985; Bossu and Feltz, 1986; Dupont et al., 1986). Interestingly, in these seminal studies, the native T-type current was mostly related to Cav3.2 (nickel-sensitive) channels (Carbone and Lux, 1984; Fedulova et al., 1985; Bean, 1985; Bossu et al., 1985; Bossu and Feltz, 1986; Dupont et al., 1986), for which we show that the intrinsic electrophysiological properties do not allow the triggering of the Ca²⁺-dependent current inhibition, contrary to Cav3.1 and Cav3.3 channels. However, and consistent with our present findings, these early studies performed in the presence of an increasing amount of [Ca²⁺]_INT have reported important features of the T-type current: (1) at 10⁻⁸ M, the T-type current was stable during 60 min whereas at 10⁻⁷ M the T-type current decreased and was suppressed after 10–15 min (Bossu et al., 1985); (2) at 10⁻⁷ M, an hyperpolarizing shift (~10 mV) of the steady-state inactivation occurred (Bossu and Feltz, 1986); and (3) at 10⁻⁶ M no T-type current was recorded at HPs above −100 mV (Dupont et al., 1986). Altogether, these historical results and our present data are in favor of a ‘minute scale’ Ca²⁺-dependent modulation of the T-type current, which induces a hyperpolarizing shift in the steady-state inactivation and consequently a decrease in T-type current amplitude (see Figure 7).

It is also important to depict our results in the light of recent findings obtained on L-type channels using high frequency of stimulation (Oliveria et al., 2007, 2012). In 15 mM external Ca²⁺, the Cav1.2 current dropped to 40% of the control amplitude during 1 Hz stimulation and this inhibition was abolished in the presence of intracellular BAPTA (Oliveria et al., 2007, 2012). The Cav1.2 current decrease was stable after 3–5 min, similar to that obtained with 40–50 s stimulation for Cav3.3 in the presence of the physiological 2 mM Ca²⁺ concentration, and the recovery was almost total after 4–5 min. However, contrary to Cav3 currents, the decrease in the Cav1.2 current stimulated at 1 Hz did not involve a shift in its steady-state inactivation (Oliveria et al., 2007, 2012), suggesting distinct mechanisms for the L-type and the T-type current modulation. In addition, a prolonged stimulation of Cav1.2 current (in 10 mM external Ca²⁺ saline) induced channel endocytosis (Green et al., 2007; Tsuruta et al., 2009; Hall et al., 2013) and, in this case, the recovery of the L-type current took approximately 30 min (Green et al., 2007). This Ca²⁺-dependent modulation of L-type channels appears distinct of that described here for Cav3 channels, both regarding the time of the recovery and the shift in the steady-state inactivation. In addition, we found that ATP treatment in cells expressing both the P2X4 receptors and the Cav3.3 channels did not induce significant changes in membrane expression of Cav3.3, suggesting that the current decrease did not involve an endocytosis mechanism. Therefore, although the L- and the T-type channel regulation share apparent similar properties, modulation of the T-type current by cumulative Ca²⁺ entry has unique features, depending mostly on the shift of the steady-state inactivation, i.e. the modulation of Cav3 availability.

The discovery of a Ca²⁺-driven feedback regulation of T-type channels may have important physiological and pathophysiological implications. Indeed, an increase in the activity of T-type channels have been implicated in several diseases linked to altered Ca²⁺ signaling (Orestes et al., 2013; Jagodic et al., 2007; Scholl et al., 2015; Zamponi, 2016; Daniil et al., 2016) and T-type channel activity is also linked to Ca²⁺ overload toxicity occurring in ischemia (Nikonenko et al., 2005; Bancila et al., 2011; Gouriou et al., 2013). Also, our study reveals that activation of Ca²⁺-permeable ionotropic receptors could also markedly inhibit T-type channel activity, and interestingly, cross-talk between these receptors and T-type channels have been recently observed (Comunanza et al., 2011; Kerckhove et al., 2014; Wang et al., 2015; Tatsuki et al., 2016).

In summary, we have identified a novel regulation pathway for T-type Ca²⁺ channels. By demonstrating that Ca²⁺ entry exerts a feedback control on T-type channel activity, our study opens up new horizons towards deciphering how this local and dynamic Ca²⁺-dependent modulation of Cav3 channels can impact the cellular and physiological roles of T-type channels in normal and disease states.

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Author details

1. Magali Cazade

   1. IGF, CNRS, INSERM, University of Montpellier, Montpellier, France
   2. LabEx 'Ion Channel Science and Therapeutics', Montpellier, France

   Contribution

   MC, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

2. Isabelle Bidaud

   1. IGF, CNRS, INSERM, University of Montpellier, Montpellier, France
   2. LabEx 'Ion Channel Science and Therapeutics', Montpellier, France

   Contribution

   IB, Investigation

   Competing interests

   The authors declare that no competing interests exist.

3. Philippe Lory

   1. IGF, CNRS, INSERM, University of Montpellier, Montpellier, France
   2. LabEx 'Ion Channel Science and Therapeutics', Montpellier, France

   Contribution

   PL, Writing—review and editing

   For correspondence

   philippe.lory@igf.cnrs.fr

   Competing interests

   The authors declare that no competing interests exist.

4. Jean Chemin

   1. IGF, CNRS, INSERM, University of Montpellier, Montpellier, France
   2. LabEx 'Ion Channel Science and Therapeutics', Montpellier, France

   Contribution

   JC, Conceptualization, Formal analysis, Supervision, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   jean.chemin@igf.cnrs.fr

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6089-5964

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