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.     

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.     

Short- and long-term roles of phosphatidylinositol 4,5-bisphosphate PIP2 on Cav3.1- and Cav3.2-T-type calcium channel current

T-type calcium (Ca²⁺) channels play important physiological functions in excitable cells including cardiomyocyte. Phosphatidylinositol-4,5-bisphosphate (PIP₂) has recently been reported to modulate various ion channels’ function. However the actions of PIP₂ on the T-type Ca²⁺ channel remain unclear. To elucidate possible effects of PIP₂ on the T-type Ca²⁺ channel, we applied patch clamp method to investigate recombinant Ca_V3.1- and Ca_V3.2-T-type Ca²⁺ channels expressed in mammalian cell lines with PIP₂ in acute- and long-term potentiation. Short- and long-term potentiation of PIP₂ shifted the activation and the steady-state inactivation curve toward the hyperpolarization direction of Ca_V3.1-I_Ca.T without affecting the maximum inward current density. Short- and long-term potentiation of PIP₂ also shifted the activation curve toward the hyperpolarization direction of Ca_V3.2-I_Ca.T without affecting the maximum inward current density. Conversely, long-term but not short-term potentiation of PIP₂ shifted the steady-state inactivation curve toward the hyperpolarization direction of Ca_V3.2-I_Ca.T. Long-term but not short-term potentiation of PIP₂ blunted the voltage-dependency of current decay Ca_V3.1-I_Ca.T. PIP₂ modulates Ca_V3.1- and Ca_V3.2-I_Ca.T not by their current density but by their channel gating properties possibly through its membrane-delimited actions.     

Modulation of T-type calcium channels by bioactive lipids

T-type calcium channels (T-channels/Ca_V3) 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 Ca_V3.1, Ca_V3.2, and Ca_V3.3 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.     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.     

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.     

Biophysical properties, pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(V)1.3) voltage-dependent calcium currents

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha(1) subunit together with several accessory subunits, including alpha(2)delta, beta, and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from alpha(1S) (skeletal muscle), alpha(1C) (in heart and brain), alpha(1D) (mainly in brain, heart, and endocrine tissue), and alpha(1F) (retina). Neuroendocrine L-type currents have a significant role in the control of neurosecretion and can be inhibited by GTP-binding (G-) proteins. However, the subunit composition of the VDCCs underlying these G-protein-regulated neuroendocrine L-type currents is unknown. To investigate the biophysical and pharmacological properties and role of G-protein modulation of alpha(1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha(1D) subunit, co-expressed with alpha(2)delta-1 and beta(3a), stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The alpha(1D)-expressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba2+) and showed little inactivation in external Ba2+, while displaying rapid inactivation kinetics in external Ca2+. The L-type currents were inhibited by the 1,4 dihydropyridine (DHP) antagonists nifedipine and nicardipine and were enhanced by the DHP agonist BayK S-(-)8644. However, alpha(1D) L-type currents were not modulated by activation of a number of G-protein pathways. Activation of endogenous somatostatin receptor subtype 2 (sst2) by somatostatin-14 or activation of transiently transfected rat D2 dopamine receptors (rD2(long)) by quinpirole had no effect. Direct activation of G-proteins by the nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also had no effect on the alpha(1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha(1B)/alpha(2)delta-1/beta(3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha(1D) clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind Gbetagamma, unlike the alpha(1B) I-II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human alpha(1D) L-type currents are similar to alpha(1C) currents, and these currents are also resistant to modulation by G(i/o)-linked G-protein-coupled receptors.     

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.     

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.     

The voltage dependence of gating currents of the neuronal CA(v)3.3 channel is determined by the gating brake in the I-II loop

Low-voltage-activated Ca_V3 Ca²⁺ channels have an activation threshold around ?60?mV, which is lower than the activation threshold of other voltage-dependent calcium channels (VDCCs). The kinetics of their activation at membrane voltages just above the activation threshold is much slower than the activation kinetics of other VDCCs. It was demonstrated recently that the intracellular loop connecting repeats I and II of all three Ca_V3 channels contains a so-called gating brake. Disruption of this brake yields channels that activate at even more hyperpolarized potentials with significantly accelerated kinetics. We have compared gating of a wild-type Ca_V3.3 channel and a mutated ID12 channel, in which the putative gating brake at the proximal part of the I?CII loop was removed. Voltage dependence of the gating current activation was shifted by 34.6?mV towards more hyperpolarized potentials in ID12 channel. ON-charge movement was significantly faster in the ID12 channel, while the kinetics of the off-charge was not altered by the mutation. We conclude that the putative gating brake in I?CII loop hinders not only the opening of the conducting pore but also the activating movement of voltage-sensing S4 segments, stabilizing the channel in its closed state.     

The dihydropyridine niguldipine inhibits T-type Ca2+ currents in atrial myocytes

The whole-cell tight seal recording technique was used to investigate the effects of niguldipine, a novel dihydropyridine, on Ca²⁺ currents in guinea pig atrial cells. Ca²⁺ currents were separated into T-type and L-type components by an appropriate voltage protocol. Extracellular application of 1 M (±)-niguldipine (NIG) resulted in a pronounced blockade of both T-type (to 20±10 % of control, n=5) and L-type Ca²⁺ currents (to 28±12 % of control, n=5). Current to voltage relationships clearly showed that both Ca²⁺ currents were blocked over the whole voltage range examined (-60 to +40 mV). The inhibitory effect of niguldipine on T-type Ca²⁺ currents was found to be voltage-dependent, i. e. prolonged hyperpolarization to -90 mV led to a partial and transient removal of NIG block. The IC₅₀ for T-type Ca²⁺ current inhibition by (±)-NIG was determined as 0.18 M. NIG action is stereospecific. (+)-niguldipine was found to be more potent than (-)-niguldipine in blocking both Ca²⁺ currents. This study demonstrates the Ca²⁺ antagonistic action of the dihydropyridine NIG, which may not discriminate between T- and L-type Ca²⁺ channels.     

T-type channel-mediated neurotransmitter release

Besides controlling a wide variety of cell functions, T-type channels have been shown to regulate neurotransmitter release in peripheral and central synapses and neuroendocrine cells. Growing evidence over the last 10 years suggests a key role of Cav3.2 and Cav3.1 channels in controlling basal neurosecretion near resting conditions and sustained release during mild stimulations. In some cases, the contribution of low-voltage-activated (LVA) channels is not directly evident but requires either the activation of coupled presynaptic receptors, block of ion channels, or chelation of metal ions. Concerning the coupling to the secretory machinery, T-type channels appear loosely coupled to neurotransmitter and hormone release. In neurons, Cav3.2 and Cav3.1 channels mainly control the asynchronous appearance of “minis” [miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory postsynaptic currents (mEPSCs)]. The same loose coupling is evident from membrane capacity and amperometric recordings in chromaffin cells and melanotropes where the low-threshold-driven exocytosis possesses the same linear Ca²⁺ dependence of the other voltage-gated Ca²⁺ channels (Cav1 and Cav2) that is strongly attenuated by slow calcium buffers. The intriguing issue is that, despite not expressing a consensus “synprint” site, Cav3.2 channels do interact with syntaxin 1A and SNAP-25 and, thus, may form nanodomains with secretory vesicles that can be regulated at low voltages. In this review, we discuss all the past and recent issues related to T-type channel-secretion coupling in neurons and neuroendocrine cells.     

Extracellular matrix proteins as substrate modulate the pattern of calcium channel expression in cultured rat retinal pigment epithelial cells

We investigated the effect of different culture substrates on the expression of membrane conductances for calcium in cultured rat retinal pigment epithelial (RPE) cells using the perforated patch technique and barium as charge carrier. In younger cultures (up to 12 days old) the RPE cells expressed L-type calcium channels, in older cultures (more than 12 days old) LVA-type channels. The LVA-type channels have been characterized as a tetrodotoxin sensitive Ca²⁺ channels. Coating the culture substrate with laminin, shifted the culture age for expression of LVA-type channels to 7 days. When collagen type 4 was used as substrate LVA-type channels and L-type channels were expressed simultaneously in 7 days old cultures. We concluded that proteins of the extracellular matrix which are known to enhance cell differentiation in culture, enhance the expression of LVA-type channels in RPE cells.     

T-type Ca2+ channels in spermatogenic cells and sperm

Cell function is importantly regulated by the intracellular concentration of Ca²⁺ ([Ca²⁺]i). Sperm development and function are deeply influenced by [Ca²⁺]i which is modulated amongst other ion transporters by plasma membrane Ca²⁺ permeable channels. The presence and role of voltage-dependent Ca²⁺ channels (Ca_V) of the T-type (Ca_V3) in sperm physiology have become a matter of debate in recent years. Though they are functionally present in later stages of development in spermatogenic cells and testicular sperm and their mRNAs and proteins detected from spermatogenic cells to mature mammalian spermatozoa, their currents have not been recorded in mature spermatozoa. This review critically summarizes the evidence for the involvement of Ca_V3 channels in sperm development and function.     

Angiotensin II regulates endothelial cell migration through calcium influx via T-type calcium channel in human umbilical vein endothelial cells

Aim: The T-type calcium channel is expressed in vascular endothelial cells, but its role in endothelial cell function is yet to be elucidated. We analysed the endothelial functional role of T-type calcium channel-dependent calcium under angiotensin II (Ang II) stimulation. Methods: Human umbilical vein endothelial cells were co-incubated with hormone at 10⁻⁷ m and either Efonidipine 10⁻⁵ m or Verapamil 10⁻⁵ m or Mibefradil 10⁻⁵ m or Wortmannin 10⁻⁶ m . The contribution of Ang II receptors was evaluated using PD123319 10⁻⁷ m and ZD 7155 10⁻⁷ m . The calcium ion concentration was observed using Fluo-3 acetossimetil ester. The cells were observed after 3, 6, 9 and 12 h. Results: The microfluorescence method points out that Ang II induces intracellular calcium modulation in time by distinct mechanisms. AT2 receptor blockade is necessary to observe significant increase in [Ca²⁺]_i levels. Pre-treatment with Mibefradil abolishes Ang II -induced cell migration. Conclusions: Our data show that Ang II, via AT1 receptor, modulates calcium concentration involving T-type calcium channel and L-type calcium channel but only the calcium influx via T-type calcium channels regulates endothelial cell migration which is essential for angiogenesis.     

Cysteines in the loop between IS5 and the pore helix of CaV3.1 are essential for channel gating

The role of six cysteines of Ca_V3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in Ca_V3.1 gating. We hypothesize that cysteines in the large extracellular loop of Ca_V3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.     

State-dependent signaling by Cav1.2 regulates hair follicle stem cell function

The signals regulating stem cell activation during tissue regeneration remain poorly understood. We investigated the baldness associated with mutations in the voltage-gated calcium channel (VGCC) Ca_v1.2 underlying Timothy syndrome (TS). While hair follicle stem cells express Ca_v1.2, they lack detectable voltage-dependent calcium currents. Ca_v1.2^TS acts in a dominant-negative manner to markedly delay anagen, while L-type channel blockers act through Ca_v1.2 to induce anagen and overcome the TS phenotype. Ca_v1.2 regulates production of the bulge-derived BMP inhibitor follistatin-like1 (Fstl1), derepressing stem cell quiescence. Our findings show how channels act in nonexcitable tissues to regulate stem cells and may lead to novel therapeutics for tissue regeneration.     

Localization of L-type calcium channel CaV1.3 in cat lumbar spinal cord – with emphasis on motoneurons

Voltage-dependent persistent inward currents (PICs) which underlie the plateau potentials are an important intrinsic property of spinal motoneurons. Electrophysiological experiments have indicated that a subtype of the low threshold L-type calcium channel, Ca_V1.3, mediates this current. In mouse and turtle lumbar spinal cord it has been shown that these channel proteins are mainly found on motoneuron dendrites. In the present study we have used immunohistochemistry to locate these channels in lumbar spinal neurons, especially motoneurons, of the cat. The results indicate that Ca_V1.3 immunoreactivity was unevenly distributed among the laminae of the spinal grey matter. The small neurons in superficial dorsal horn (laminae I–III) were sparsely and weakly labelled, while large neurons in ventral horn were frequently and densely labelled. Groups of motoneurons in lamina IX that were immunoreactive to choline acetyltransferase also co-expressed Ca_V1.3. The immmunoreactivity was mainly associated with neuronal somata and proximal dendrites. Double staining with antibodies against Ca_V1.3 and MAP2 (a dendritic marker) showed that some fine fibres, which may include distal dendrites, were also labelled. These results in the cat spinal cord show some differences from studies in mouse and turtle motoneurons where the immunoreactivity against this channel was mainly localized to the dendrites.     

5,6-EET potently inhibits T-type calcium channels: implication in the regulation of the vascular tone

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_v3.1, Ca_v3.2, and Ca_v3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Ca_v3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC₅₀?=?0.54 μM), occurred in the minute range, and were reversible. The 5,6-EET inhibited the Ca_v3 currents at physiological resting membrane potentials mostly by inducing a large negative shift in their steady-state inactivation properties. Using knockout mice for Ca_v3.1 and Ca_v3.2, we demonstrated that the vasodilation of preconstricted mesenteric arteries induced by 5,6-EET was specifically impaired in Ca_v3.2 knockout mice. Overall, our results indicate that inhibition of Ca_v3 currents by 5,6-EET is an important mechanism controlling the vascular tone.