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Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca2+ channels. We demonstrate that a rise in submembrane Ca2+ induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca2+-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca2+ entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca2+ channels to their physiological roles. DOI: http://dx.doi.org/10.7554/eLife.22331.001, eLife digest 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. DOI: http://dx.doi.org/10.7554/eLife.22331.002

T-type (Cav3) calcium channels play important roles in neuronal excitability, both in normal and pathological activities of the brain. In particular, they contribute to hyper-excitability disorders such as epilepsy. Here we have characterized the anticonvulsant properties of TTA-A2, a selective T-type channel blocker, in mouse. Using the maximal electroshock seizure (MES) as a model of tonic-clonic generalized seizures, we report that mice treated with TTA-A2 (0.3 mg/kg and higher doses) were significantly protected against tonic seizures. Although no major change in Local Field Potential (LFP) pattern was observed during the MES seizure, analysis of the late post-ictal period revealed a significant increase in the delta frequency power in animals treated with TTA-A2. Similar results were obtained for Cav3.1-/- mice, which were less prone to develop tonic seizures in the MES test, but not for Cav3.2-/- mice. Analysis of extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and c-Fos expression revealed a rapid and elevated neuronal activation in the hippocampus following MES clonic seizures, which was unchanged in TTA-A2 treated animals. Overall, our data indicate that TTA-A2 is a potent anticonvulsant and that the Cav3.1 isoform plays a prominent role in mediating TTA-A2 tonic seizure protection.

T-type calcium channels (T-channels) contribute to a wide variety of physiological functions, especially in the cardiovascular and nervous systems. Recent studies using knock-out mouse models have been instrumental in documenting further the role of T-channels in sleep, heartbeat, pain and epilepsy. Importantly, several novel aspects of the regulation of these channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Here, we review recent evidence supporting that the Cav3 subunits of T-channels are modulated by endogenous ligands such as anandamide, zinc, redox and oxidizing agents, as well as G-protein and protein kinases pathways. The study of T-channel mutations associated with childhood absence epilepsy has also revealed new aspects of Cav3 subunit trafficking. Collectively, these findings identify novel regulatory mechanisms involved in the fine tuning of T-channel expression and activity, and offer new directions for the design of novel therapeutic strategies targeting these channels.

Modulation of low voltage-activated Ca(V)3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca(V)1 and Ca(V)2 calcium channels. Notably, it is yet unresolved whether Ca(V)3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature ( approximately 30-37 degrees C) but not at room temperature ( approximately 22-27 degrees C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca(V)2.3 currents. We also report that neither Ca(V)3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca(V)3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca(V)3 T-channels and further highlight the key role of the physiological temperature in the effects described.

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.

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 (CaV3.1, CaV3.2 and CaV3.3). Using tsA-201 cells, we demonstrate that CaV3.2 current (IC50, 0.8 μm) is significantly more sensitive to Zn2+ than are CaV3.1 and CaV3.3 currents (IC50, 80 μm and ∼160 μm, respectively). This inhibition of CaV3 currents is associated with a shift to more negative membrane potentials of both steady-state inactivation for CaV3.1, CaV3.2 and CaV3.3 and steady-state activation for CaV3.1 and CaV3.3 currents. We also document changes in kinetics, especially a significant slowing of the inactivation kinetics for CaV3.1 and CaV3.3, but not for CaV3.2 currents. Notably, deactivation kinetics are significantly slowed for CaV3.3 current (∼100-fold), but not for CaV3.1 and CaV3.2 currents. Consequently, application of Zn2+ results in a significant increase in CaV3.3 current in action potential clamp experiments, while CaV3.1 and CaV3.2 currents are significantly reduced. In neuroblastoma NG 108-15 cells, the duration of CaV3.3-mediated action potentials is increased upon Zn2+ application, indicating further that Zn2+ behaves as a CaV3.3 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.

Low-voltage-activated T-type calcium channels are expressed in various tissues, especially in the brain, where they promote neuronal firing and are involved in slow wave sleep and absence epilepsy. While the transduction pathways by which hormones and neurotransmitters modulate high-voltage-activated calcium channels are beginning to be unraveled, those implicated in T-type calcium channel regulation remain obscure. Several neurotransmitters and hormones regulate native T-type calcium channels, although some contradictory data have been reported depending on the cell type studied. This review focuses on the short-term (minutes range) modulation of T-type calcium channels by neurotransmitters and hormones and on the roles of G proteins and protein kinases in these modulatory effects. Results obtained in different native tissues are discussed and compared with the more recent studies of the three cloned T-type calcium channels CaV3.1, CaV3.2 and CaV3.3 in expression systems.

Calcium influx via low-voltage activated alpha(1H) (Ca(v)3.2) T-currents participates in the morphological and electrical differentiation of neuroblastoma NG108-15 cells. We investigated whether an autocrine mechanism could contribute to this differentiation process. The presence of factors secreted by NG108-15 cells was identified through the use of conditioned media (CM) obtained from differentiated cells. These CM significantly increased neuritogenesis with no change in the HVA calcium channel expression. CM-induced neuritogenesis persists during alpha(1H) current block, whereas CM obtained from cells transfected with an alpha(1H) antisense did not induce neuritogenesis. These data indicate that morphological differentiation of NG108-15 cells depends on an autocrine mechanism, which is controlled by alpha(1H) currents. Such a mechanism is likely to play a role in the various differentiation processes that imply alpha(1H) T-type Ca(2+) channels.

Neuronal differentiation involves both morphological and electrophysiological changes, which depend on calcium influx. Voltage-gated calcium channels (VGCCs) represent a major route for calcium entry into neurons. The recently cloned low-voltage-activated T-type calcium channels (T-channels) are the first class of VGCCs functionally expressed in most developing neurons, as well as in neuroblastoma cell lines, but their roles in neuronal development are yet unknown. Here, we document the part played by T-channels in neuronal differentiation. Using NG108-15, a cell line that recapitulates early steps of neuronal differentiation, we demonstrate that blocking T-currents by nickel, mibefradil, or the endogenous cannabinoid anandamide prevents neuritogenesis without affecting neurite outgrowth. Similar results were obtained using antisense oligodeoxynucleotides directed against the alpha1H T-channel subunit. Furthermore, we describe that inhibition of alpha1H T-channel activity impairs concomitantly, but independently, both high-voltage-activated calcium channel expression and neuritogenesis, providing strong evidence for a dual role of T-channels in both morphological and electrical changes at early stages of neuronal differentiation.

In several types of neurons, firing is an intrinsic property produced by specific classes of ion channels. Low-voltage-activated T-type calcium channels (T-channels), which activate with small membrane depolarizations, can generate burst firing and pacemaker activity. Here we have investigated the specific contribution to neuronal excitability of cloned human T-channel subunits. Using HEK-293 cells transiently transfected with the human alpha(1G) (Ca(V)3.1), alpha(1H) (Ca(V)3.2) and alpha(1I) (Ca(V)3.3) subunits, we describe significant differences among these isotypes in their biophysical properties, which are highlighted in action potential clamp studies. Firing activities occurring in cerebellar Purkinje neurons and in thalamocortical relay neurons used as voltage clamp waveforms revealed that alpha(1G) channels and, to a lesser extent, alpha(1H) channels produced large and transient currents, while currents related to alpha(1I) channels exhibited facilitation and produced a sustained calcium entry associated with the depolarizing after-potential interval. Using simulations of reticular and relay thalamic neuron activities, we show that alpha(1I) currents contributed to sustained electrical activities, while alpha(1G) and alpha(1H) currents generated short burst firing. Modelling experiments with the NEURON model further revealed that the alpha(1G) channel and alpha(1I) channel parameters best accounted for T-channel activities described in thalamocortical relay neurons and in reticular neurons, respectively. Altogether, the data provide evidence for a role of alpha(1I) channel in pacemaker activity and further demonstrate that each T-channel pore-forming subunit displays specific gating properties that account for its unique contribution to neuronal firing.

T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation via its metabolism leading to prostanoids, leukotrienes, and epoxyeicosatrienoic acids (EETs). However, the effects of these latter molecules on T-currents have not been investigated. Here, we describe the effects of the major cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products on the three human recombinant T-channels (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Ca(v)3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC50 = 0.54 mu M), occurred in the minute range, and were reversible. The 5,6-EET inhibited the Ca(v)3 currents at physiological resting membrane potentials mostly by inducing a large negative shift in their steady-state inactivation properties. Using knockout mice for Ca(v)3.1 and Ca(v)3.2, we demonstrated that the vasodilation of preconstricted mesenteric arteries induced by 5,6-EET was specifically impaired in Ca(v)3.2 knockout mice. Overall, our results indicate that inhibition of Ca(v)3 currents by 5,6-EET is an important mechanism controlling the vascular tone.

Low-voltage-activated or T-type Ca(2+) channels (T-channels) are widely expressed, especially in the central nervous system where they contribute to pacemaker activities and are involved in the pathogenesis of epilepsy. Proper elucidation of their cellular functions has been hampered by the lack of selective pharmacology as well as the absence of generic endogenous regulations. We report here that both cloned (alpha(1G), alpha(1H) and alpha(1I) subunits) and native T-channels are blocked by the endogenous cannabinoid, anandamide. Anandamide, known to exert its physiological effects through cannabinoid receptors, inhibits T-currents independently from the activation of CB1/CB2 receptors, G-proteins, phospholipases and protein kinase pathways. Anandamide appears to be the first endogenous ligand acting directly on T-channels at submicromolar concentrations. Block of anandamide membrane transport by AM404 prevents T-current inhibition, suggesting that anandamide acts intracellularly. Anandamide preferentially binds and stabilizes T-channels in the inactivated state and is responsible for a significant decrease of T-currents associated with neuronal firing activities. Our data demonstrate that anandamide inhibition of T-channels can regulate neuronal excitability and account for CB receptor-independent effects of this signaling molecule.

The recently cloned T-type calcium channel alpha1I (Cav3.3) displays atypically slow kinetics when compared to native T-channels. Possible explanations might involve alternative splicing of the alpha1I subunit, or the use of expression systems that do not provide a suitable environment (auxiliary subunit, phosphorylation, glycosylation...). In this study, two human alpha1I splice variants, the alpha1I-a and alpha1I-b isoforms that harbour distinct carboxy-terminal regions were studied using various expression systems. As the localization of the alpha1I subunit is primarily restricted to neuronal tissues, its functional expression was conducted in the neuroblastoma/glioma cell line NG 108-15, and the results compared to those obtained in HEK-293 cells and Xenopus oocytes. In Xenopus oocytes, both isoforms exhibited very slow current kinetics compared to those obtained in HEK-293 cells, but the alpha1I-b isoform generated faster currents than the alpha1I-a isoform. Both activation and inactivation kinetics of alpha1I currents were significantly faster in NG 108-15 cells, while deactivating tail currents were two times slower, compared to those obtained in HEK-293 cells. Moreover, the alpha1-b isoform showed significantly slower deactivation kinetics both in NG 1080-15 and in HEK-293 cells. Altogether, these data emphasize the advantage of combining several expression systems to reveal subtle differences in channel properties and further indicate that the major functional differences between both human alpha1I isoforms are related to current kinetics. More importantly, these data suggest that the expression of the alpha1I subunit in neuronal cells contributes to the "normalization" of current kinetics to the more classical, fast-gated T-type Ca2+ current.

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.

T-type calcium channels (T-channels/CaV3) have unique biophysical properties allowing a calcium influx at resting membrane potential of most cells. T-channels are ubiquitously expressed in many tissues and contribute to low-threshold spikes and burst firing in central neurons as well as to pacemaker activities in cardiac cells. They also emerged as potential targets to treat cancer and hypertension. Regulation of these channels appears complex, and several studies have indicated that CaV3.1, CaV3.2, and CaV3.3 currents are directly inhibited by multiple endogenous lipids independently of membrane receptors or intracellular pathways. These bioactive lipids include arachidonic acid and ω3 poly-unsaturated fatty acids; the endocannabinoid anandamide and other N-acylethanolamides; the lipoamino-acids and lipo-neurotransmitters; the P450 epoxygenase metabolite 5,6-epoxyeicosatrienoic acid; as well as similar molecules with 18–22 carbons in the alkyl chain. In this review, we summarize evidence for direct effects of these signaling molecules, the molecular mechanisms underlying the current inhibition, and the involved chemical features. The impact of this modulation in physiology and pathophysiology is discussed with a special emphasis on pain aspects and vasodilation. Overall, these data clearly indicate that T-current inhibition is an important mechanism by which bioactive lipids mediate their physiological functions.

T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the ω3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy)pyridin-2-yl)ethyl)acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [(3)H]TTA-A1 [(R)-2-(4-(tert-butyl)phenyl)-N-(1-(5-methoxypyridin-2-yl)ethyl)acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-l-serine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [(3)H]TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with Ki in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [(3)H]TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl)benzonitrile], a positive allosteric modulator of [(3)H]TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca(v)3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.

T-type calcium channels, i.e. Cav3.1, Cav3.2 and Cav3.3 channels, have important roles in cell excitability and calcium signalling and contribute to a wide variety of physiological functions especially in nervous system. Over the past few years, several endogenous ligands regulating Cav3 activity were identified, including bioactive lipids such as the endocannabinoid anandamide (N-arachidonoyl ethanolamine). We now provide evidence that the T-type / Cav3 calcium channels are potently and reversibly inhibited by various lipoamino acids, including N-arachidonoyl glycine (NAGly, IC50 ∼ 600 nM for Cav3.2) and N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH, IC50 ∼200 nM for Cav3.2). This inhibition involves a large shift in the Cav3.2 steady-state inactivation and persists during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. It appears that lipoamino acids are the most active endogenous ligand family acting on T-channels. Importantly, lipoamino acids have weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents as well as on TRPV1 and TASK1 currents. These data indicate that lipoamino acid effects may be selective of T-type channels over HVA calcium channels, sodium channels as well as the anandamide-sensitive TRPV1 and TASK1 channels. It also suggests that these ligands can modulate multiple cell functions via T-type calcium channel regulation. In line with this, we found that lipoamino acids evoke a thermal analgesia in wild-type but not in Cav3.2 KO mice. Collectively, our data identify lipoamino acids as a new potent and selective family of endogenous T-type channel inhibitors.

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, includingN-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC50∼200 nmforN-arachidonoyl 3-OH-γ-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.

Genomic mining, the process of sifting through billions of genomic and EST sequences of several different species has led to the molecular identification of a family of low voltage activating channels, more commonly referred to as T-type channels. Historically, these channels were initially identified through the use of the patch-damp technique on various neuronal preparations. They were characterized by their small conductance, rapid voltage-dependent inactivation, a small window current and slow deactivation kinetics and their remarkable property of being able to open at membrane potentials just above the resting membrane potential of neurons. This property would allow for the entry of Ca2+ without the initiation of an action potential triggered by the opening of sodium channels. Thus the activity of these channels would contribute to modifying membrane excitability, allowing Ca2+ signaling events to occur at subthreshold potentials, and potentially modulate waveform patterns.

Low-voltage activated, T-type calcium channels (T-channels) are expressed in many developing tissues and may be important in regulating important cellular phenotype transitions leading to cell proliferation, differentiation, growth and death. The purpose of this review is to relate and delineate the current data on the involvement of T-channels in differentiation and proliferation. Owing to the recent cloning of the CaV3.1, CaV3.2 and CaV3.3 subunits coding for T-channels, classical electrophysiological and pharmacological approaches are now being supported by molecular investigations. As T-channels are expressed in early development as well as re-expressed in several disease-states, our goal is to provide a comprehensive scheme of the current hypothesis connecting the activity of T-channels to cell differentiation and proliferation, as well as the potential physiological and pathophysiological implications.

Fluoxetine, a widely used antidepressant that primarily acts as a selective serotonin reuptake inhibitor, also inhibits various neuronal ion channels. Using the whole-cell patch-clamp technique, we have examined the effects of fluoxetine and norfluoxetine, its major active metabolite, on cloned low-voltage-activated T-type calcium channels (T channels) expressed in tsA 201 cells. Fluoxetine inhibited the three T channels Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 in a concentration-dependent manner (IC(50) = 14, 16, and 30 microM, respectively). Norfluoxetine was a more potent inhibitor than fluoxetine, especially on the Ca(V)3.3 T current (IC(50) = 5 microM). The fluoxetine block of T channels was voltage-dependent because it was significantly enhanced for T channels in the inactivated state. Fluoxetine caused a hyperpolarizing shift in steady-state inactivation, with a slower rate of recovery from the inactivated state. These results indicated a tighter binding of fluoxetine to the inactivated state than to the resting state of T channels, suggesting a more potent inhibition of T channels at physiological resting membrane potential. Indeed, fluoxetine and norfluoxetine at 1 microM strongly inhibited cloned T currents (approximately 50 and approximately 75%, respectively) in action potential clamp experiments performed with firing activities of thalamocortical relay neurons. Altogether, these data demonstrate that clinically relevant concentrations of fluoxetine exert a voltage-dependent block of T channels that may contribute to this antidepressant's pharmacological effects.

International audience; Cellular or chemical activators for most transient receptor potential channels of the vanilloid subfamily (TRPV) have been identified in recent years. A remarkable exception to this is TRPV2, for which cellular events leading to channel activation are still a matter of debate. Diverse stimuli such as extreme heat or phosphatidylinositol-3 kinase (PI3-kinase) regulated membrane insertion have been shown to promote TRPV2 channel activity. However, some of these results have proved difficult to reproduce and may underlie different gating mechanisms depending on the cell type in which TRPV2 channels are expressed. Here, we show that expression of recombinant TRPV2 can induce cytotoxicity that is directly related to channel activity since it can be prevented by introducing a charge substitution in the pore-forming domain of the channel, or by reducing extracellular calcium. In stably transfected cells, TRPV2 expression results in an outwardly rectifying current that can be recorded at all potentials, and in an increase of resting intracellular calcium concentration that can be partly prevented by serum starvation. Using cytotoxicity as a read-out of channel activity and direct measurements of cell surface expression of TRPV2, we show that inhibition of the PI3-kinase decreases TRPV2 channel activity but does not affect the trafficking of the channel to the plasma membrane. It is concluded that PI3-kinase induces or modulates the activity of recombinant TRPV2 channels; in contrast to the previously proposed mechanism, activation of TRPV2 channels by PI3-kinase is not due to channel translocation to the plasma membrane.

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.

We describe here several novel properties of the human alpha(1G) subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several alpha(1G) isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: alpha(1G-a) and alpha(1G-b). Northern blot and dot blot analyses indicated that alpha(1G) mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that alpha(1G) mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest alpha(1G) isoform identified to date, alpha(1G-b), resulted in transient, low threshold activated Ca(2+) currents with the expected permeability ratio (I(Sr)I(Ca)/= I(Ba)) and channel conductance ( approximately 7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca(2+) channels. This alpha(1G)-related current was inhibited by mibefradil (IC(50) = 2 microM) and weakly blocked by Ni(2+) ions (IC(50) = 148 microM) and amiloride (IC(50)1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of -65 to -55 mV. Using neuronal action potential waveforms, we show that alpha(1G) channels produce a massive and sustained Ca(2+) influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca(2+) influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca(2+) channels.