Role of T-type channels in vasomotor function: team player or chameleon?

Low-voltage-activated T-type calcium channels play an important role in regulating cellular excitability and are implicated in conditions, such as epilepsy and neuropathic pain. T-type channels, especially Cav3.1 and Cav3.2, are also expressed in the vasculature, although patch clamp studies of isolated vascular smooth muscle cells have in general failed to demonstrate these low-voltage-activated calcium currents. By contrast, the channels which are blocked by T-type channel antagonists are high-voltage activated but distinguishable from their L-type counterparts by their T-type biophysical properties and small negative shifts in activation and inactivation voltages. These changes in T-channel properties may result from vascular-specific expression of splice variants of Cav3 genes, particularly in exon 25/26 of the III–IV linker region. Recent physiological studies suggest that T-type channels make a small contribution to vascular tone at low intraluminal pressures, although the relevance of this contribution is unclear. By contrast, these channels play a larger role in vascular tone of small arterioles, which would be expected to function at lower intra-vascular pressures. Upregulation of T-type channel function following decrease in nitric oxide bioavailability and increase in oxidative stress, which occurs during cardiovascular disease, suggests that a more important role could be played by these channels in pathophysiological situations. The ability of T-type channels to be rapidly recruited to the plasma membrane, coupled with their subtype-specific localisation in signalling microdomains where they could modulate the function of calcium-dependent ion channels and pathways, provides a mechanism for rapid up- and downregulation of vasoconstriction. Future investigation into the molecules which govern these changes may illuminate novel targets for the treatment of conditions such as therapy-resistant hypertension and vasospasm.     

Surface expression and function of Cav3.2 T-type calcium channels are controlled by asparagine-linked glycosylation

Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in T-type channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Ca_v3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Ca_v3.2 channel revealed that N-linked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Ca_v3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Ca_v3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca²⁺ current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.     

T-type calcium channel blockers as neuroprotective agents

T-type calcium channels are expressed in many diverse tissues, including neuronal, cardiovascular, and endocrine. T-type calcium channels are known to play roles in the development, maintenance, and repair of these tissues but have also been implicated in disease when not properly regulated. Calcium channel blockers have been developed to treat various diseases and their use clinically is widespread due to both their efficacy as well as their safety. Aside from their established clinical applications, recent studies have suggested neuroprotective effects of T-type calcium channel blockers. Many of the current T-type calcium channel blockers could act on other molecular targets besides T-type calcium channels making it uncertain whether their neuroprotective effects are solely due to blocking of T-type calcium channels. In this review, we discuss these drugs as well as newly developed chemical compounds that are designed to be more selective for T-type calcium channels. We review in vitro and in vivo evidence of neuroprotective effects by these T-type calcium channel blockers. We conclude by discussing possible molecular mechanisms underlying the neuroprotective effects by T-type calcium channel blockers.     

T-type calcium channels mediate rebound firing in intact deep cerebellar neurons

Neurons of the deep cerebellar nuclei (DCN) form the main output of the cerebellar circuitry and thus play an important role in cerebellar motor coordination. A prominent biophysical property observed in rat DCN neurons is rebound firing; a brief but strong hyperpolarizing input transiently increases their firing rate to much higher levels compared with that prior to the inhibitory input. Low-threshold T-type voltage-gated calcium channels have been suspected for a long time to be responsible for this phenomenon, but direct pharmacological evidence in support of this proposition is lacking. Even though a multitude of functional roles has been assigned to rebound firing in DCN neurons, their prevalence under physiological conditions is in question. Studies aimed at delineating the physiological role of rebound firing are hampered by the lack of a good pharmacological blocker. Here we show that mibefradil, a compound that blocks T-type calcium channels, potently blocks rebound firing in DCN neurons. In whole-cell experiments both mibefradil and NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]. a more selective T-type calcium channel blocker, effectively blocked rebound firing produced by direct current injection. Thus, mibefradil and other T-type channel modulators may prove to be invaluable tools for elucidating the functional importance of DCN rebound firing in cerebellar computation.     

T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis

T-type channels are transient low-voltage-activated (LVA) Ca²⁺ channels that control Ca²⁺ entry in excitable cells during small depolarizations around resting potential. Studies in the past 20 years focused on the biophysical, physiological, and molecular characterization of T-type channels in most tissues. This led to a well-defined picture of the functional role of LVA channels in controlling low-threshold spikes, oscillatory cell activity, muscle contraction, hormone release, cell growth and differentiation. So far, little attention has been devoted to the role of T-type channels in transmitter release, which mainly involves channel types belonging to the high-voltage-activated (HVA) Ca²⁺ channel family. However, evidence is accumulating in favor of a unique participation of T-type channels in fast transmitter release. Clear data are now reported in reciprocal synapses of the retina and olfactory bulb, synaptic contacts between primary afferent and second order nociceptive neurons, rhythmic inhibitory interneurons of invertebrates and clonal cell lines transfected with recombinant α₁ channel subunits. T-type channels also regulate the large dense-core vesicle release of neuroendocrine cells where Ca²⁺ dependence, rate of vesicle release, and size of readily releasable pool appear comparable to those associated to HVA channels. This suggests that when sufficiently expressed and properly located near the release zones, T-type channels can trigger fast low-threshold secretion. In this study, we will review the main findings that assign a specific task to T-type channels in fast exocytosis, discussing their possible involvement in the control of the Ca²⁺-dependent processes regulating exocytosis like vesicle depletion and vesicle recycling.     

Role of voltage-gated calcium channels in epilepsy

It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high- and low-voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Ca_v2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Ca_v2.1 α1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to co-assemble with Ca_v2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits, there is a compensatory increase in thalamic LVA currents which likely leads to the seizure phenotype. In fact, gain-of-function mutations have been identified in Ca_v3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. In this paper, we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.     

T-type Ca2+ channels and the urinary and male genital tracts

T-type Ca²⁺ channels are widely expressed throughout the urinary and male genital tracts, generally alongside L-type Ca²⁺ channels. The use of pharmacological blockers of these channels has suggested functional roles in all regions, with the possible exception of the ureter. Their functional expression is apparent not just in smooth muscle cells but also in interstitial cells that lie in close proximity to muscle, nerve and epithelial components of these tissues. Thus, T-type Ca²⁺ channels can contribute directly to modulation of muscle function and indirectly to changes of epithelial and nerve function. T-type Ca²⁺ channel activity modulates phasic contractile activity, especially in conjunction with Ca²⁺-activated K⁺ channels, and also to agonist-dependent responses in different tissues. Upregulation of channel density occurs in pathological conditions associated with enhanced contractile responses, e.g. overactive bladder, but it is unclear if this is causal or a response to the pathological state. Moreover, T-type Ca²⁺ channels may have a role in the development of prostate tumours regulating the secretion of mitogens from neuroendocrine cells. Although a number of selective channel blockers exist, their relative selectivity over L-type Ca²⁺ channels is often low and makes evaluation of T-type Ca²⁺ channel function in the whole organism difficult.     

Ca2+ signaling by T-type Ca2+ channels in neurons

Among the major families of voltage-gated Ca²⁺ channels, the low-voltage-activated channels formed by the Ca_v3 subunits, referred to as T-type Ca²⁺ channels, have recently gained increased interest in terms of the intracellular Ca²⁺ signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca²⁺ channels act as an important Ca²⁺ source in a wide range of neuronal cell types. The work is focused on T-type Ca²⁺ channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca²⁺ entering through T-type Ca²⁺ channels have been described. Notably, Ca²⁺ influx through T-type Ca²⁺ channels is the predominant Ca²⁺ source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca²⁺ signaling through T-type Ca²⁺ channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions. Lucius Cueni and Marco Canepari contributed equally to this review.     

ZD 7288 inhibits T-type calcium current in rat hippocampal pyramidal cells

Many studies have used the channel blocker ZD 7288 to assess possible physiological and pathophysiological roles of hyperpolarization-activated cation currents (I_h). In view of the known interplay between I_h and other membrane conductances, the effects in Wistar rats of ZD 7288 on low-voltage-activated (LVA (− or T-type)) Ca²⁺ channels were examined in whole-cell patch-clamp recordings from CA1 pyramidal cells in the presence of TTX, TEA, 4-AP, CsCl, BaCl₂ and nifedipine. ZD 7288 reduced T-type calcium channel currents and this effect was concentration dependant. ZD 7288 blocked T-type currents when applied extracellularly, but not when included in the recording pipette. Furthermore, ZD 7288 altered the steady-state voltage-dependent inactivation of T-currents. These results indicate that the blocker ZD 7288 has effects on voltage sensitive channels additional to those reported for the I_h current.     

Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve

Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate density (20 +/- 2 pA/pF). In rats with CCI, T-type current density (30 +/- 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacological sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our finding that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacological and molecular studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain associated with partial mechanical injury to the sciatic nerve.     

Neuropathic pain: role for presynaptic T-type channels in nociceptive signaling

Pain is an important clinical problem and, in its chronic form, may be a disabling condition. Most currently available therapies are insufficient and/or accompanied by serious side effects. Recent studies have implicated the Ca_V3.2 isoform of T-type Ca channels in nociceptive signaling. Ca_V3.2 channels are located in the somas of dorsal root ganglion cells and in the central endings of these cells in the dorsal horn of the spinal cord. These channels can support the development and maintenance of both physiological (nociceptive) and pathological (neuropathic) pain. In this review, we summarize the most recent evidence linking the presynaptic Ca_V3.2 channels to the etiology of neuropathic pain disorders. In particular, we focus on data linking plasticity of Ca_V3.2 channels with neuropathic pain disorders associated with mechanical peripheral nerve injury and with diabetic peripheral neuropathy. We also discuss the development of potential pain therapies aimed at these channels.     

T-type calcium channels blockers as new tools in cancer therapies

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca²⁺ channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca²⁺ channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca²⁺ channels could be attractive molecular targets for anticancer therapy.     

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Ca_v channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Ca_v channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca²⁺ through Ca_v channels contributes to the regulation of hLEC proliferation, identifying Ca_v channel blockers as potential therapeutic substances in ocular diseases.     

Evidence for common structural determinants of activation and inactivation in T-type Ca2+ channels

One of the most distinctive features of T-type Ca²⁺ channels is their fast inactivation. Recent structure–function studies indicate that the rate of macroscopic inactivation of these channels is influenced by several structural components, including intracellular linkers, transmembrane segments, and pore loops. The macroscopic inactivation of T-type channels is partially coupled to activation. It is therefore possible that changes in the rate of macroscopic inactivation after alteration in the structure of these channels might actually result from changes in activation kinetics. In this study, we use kinetic simulations to illustrate how the alteration of the rate of channel activation may lead to changes in the rate of macroscopic inactivation. By examining data pooled from several structure–function studies we demonstrate that gating modifications induced by alteration in the channel structure unveils a correlation between the time constants of macroscopic inactivation and activation. This analysis underscores the relevance of considering the inactivation–activation coupling when analyzing the structural determinants of T-type channel inactivation. Furthermore, we demonstrate that slow-inactivating mutants, with modifications in the IIIS6 segment and the proximal C terminus, display significant alterations in the voltage dependencies of activation and deactivation with respect to the wild type channel Ca_V3.1. Our results indicate that common structures, most likely the S6 transmembrane segments, are involved in the conformational changes occurring during both channel activation and inactivation.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Regulation of firing response gain by calcium-dependent mechanisms in vestibular nucleus neurons

Behavioral reflexes can be modified by experience via mechanisms that are largely unknown. Within the circuitry for the vestibuloocular reflex (VOR), neurons in the medial vestibular nucleus (MVN) show adaptive changes in firing rate responses that are correlated with VOR gain (the ratio of evoked eye velocity to input head velocity). Although changes in synaptic strength are typically assumed to underlie gain changes in the VOR, modulation of intrinsic ion channels that dictate firing could also play a role. Little is known, however, about how ion channel function or regulation contributes to firing responses in MVN neurons. This study examined contributions of calcium-dependent currents to firing responses in MVN neurons recorded with whole cell patch electrodes in rodent brain stem slices. Firing responses were remarkably linear over a wide range of firing rates and showed modest spike frequency adaptation. Firing response gain, the ratio of evoked firing rate to input current, was reduced by increasing extracellular calcium and increased either by lowering extracellular calcium or with antagonists to SK- and BK-type calcium-dependent potassium channels and N- and T-type calcium channels. Blockade of SK channels occluded gain increases via N-type calcium channels, while blocking BK channels occluded gain increases via presumed T-type calcium channels, indicating specific coupling of potassium channels and their calcium sources. Selective inhibition of Ca(2+)/calmodulin-dependent kinase II and broad-spectrum inhibition of phosphatases modulated gain via BK-dependent pathways, indicating that firing responses are tightly regulated. Modulation of firing response gain by phosphorylation provides an attractive mechanism for adaptive control of VOR gain.     

T-type channels in the sino-atrial and atrioventricular pacemaker mechanism

Cardiac automaticity is a fundamental physiological function in vertebrates. Heart rate is under the control of several neurotransmitters and hormones and is permanently adjusted by the autonomic nervous system to match the physiological demand of the organism. Several classes of ion channels and proteins involved in intracellular Ca²⁺ handling contribute to pacemaker activity. Voltage-dependent T-type Ca²⁺ channels are an integral part of the complex mechanism underlying pacemaking. T-type channels also contribute to impulse conduction in mice and humans. Strikingly, T-type channel isoforms are co-expressed in the cardiac conduction system with other ion channels that play a major role in pacemaking such as f- (HCN4) and L-type Ca_v1.3 channels. Pharmacologic inhibition of T-type channels reduces the spontaneous activity of isolated pacemaker myocytes of the sino-atrial node, the dominant heart rhythmogenic centre. Target inactivation of T-type Ca_v3.1 channels abolishes I _Ca,T in both sino-atrial and atrioventricular myocytes and reduces the daily heart rate of freely moving mice. Ca_v3.1 channels contribute also to automaticity of the atrioventricular node and to ventricular escape rhythms, thereby stressing the importance of these channels in automaticity of the whole cardiac conduction system. Accordingly, loss-of-function of Ca_v3.1 channels contributes to severe form of congenital bradycardia and atrioventricular block in paediatric patients.     

Synthesis and Evaluation of 1,4-Dihydropyridine Derivatives with Calcium Channel Blocking Activity

1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors.     

Low-voltage-activated T-type Ca2+ channel inhibitors as new tools in the treatment of glioblastoma: the role of endostatin

Ca²⁺ plays a key role in intracellular signaling and controls various cellular processes such as proliferation, differentiation, cell growth, death, and apoptosis. Aberrant changes in intracellular Ca²⁺ levels can promote undesired cell proliferation and migration and are therefore associated with certain tumor types. Many research groups have suggested a potential role for voltage-gated Ca²⁺ channels in the regulation of tumor growth and progression, particularly T-type channels due to their unique biophysical properties. T-type channels are expressed in normal tissues throughout the body and in different types of tumors such as breast carcinoma, retinoblastoma, neuroblastoma, and glioma. It has been demonstrated that increased functional expression of the α1 subunit of T-type channels plays a role in the abnormal proliferation of glioblastoma cells. As such, siRNA-mediated knockdown of the expression of the α1 subunit of T-type channels decreases the proliferation of these cells. Moreover, pharmacological blockade of T-type channels significantly decreases tumor growth. In this review, we focus on the use of T-type channel blockers for the potential treatment of cancers, particularly highly proliferative tumors such as glioblastoma. We conclude that T-type channel blockers such as endostatin can serve as a potential therapeutic tool for tumors whose proliferation depends on increased T-type channel expression.     

CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy

Ca_V3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that Ca_V3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional Ca_V3.2 channels primarily incorporate into synapses, replace existing Ca_V3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCa_V3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCa_V3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of Ca_V3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE.