β-Adrenergic stimulation increases Cav3.1 activity in cardiac myocytes through protein kinase A

The T-type Ca(2+) channel (TTCC) plays important roles in cellular excitability and Ca(2+) regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca(2+) current (I(Ca-T(3.1))). I(Ca-T(3.1)) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased I(Ca-T(3.1)) as well as I(Ca-L) in TG-VMs at both physiological and room temperatures. The ISO effect on I(Ca-L) and I(Ca-T) in TG myocytes was blocked by H89, a PKA inhibitor. I(Ca-T) was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of I(Ca-T) in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. I(Ca-T) in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases I(Ca-T(3.1)) in cardiomyocytes(,) which is mediated by the cAMP/PKA pathway. The upregulation of I(Ca-T(3.1)) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca (2+) is essential for cardiomyocyte function, and alterations in proteins that regulate Ca (2+) influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca (2+) channels are one pathway of Ca (2+) entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Cav3.1 (α1G) and Cav3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca (2+) channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Cav3.1 and Cav3.2 Ca (2+) channel expression in the pathological setting. The putative association of T-type Ca (2+) channels with Ca (2+) dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Epigallocatechin-3-gallate elicits Ca spike in MCF-7 breast cancer cells: Essential role of Cav3.2 channels

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca²⁺ channels toward a mechanistic study on the effect of EGCG on [Ca²⁺]_i. Confocal Ca²⁺ imaging showed that EGCG induces a [Ca²⁺]_i spike which is due to extracellular Ca²⁺ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca²⁺channel blockers. siRNA knockdown of T-type Ca²⁺ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K⁺ currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K⁺ channel induced a reduction of the EGCG [Ca²⁺]_i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K⁺ current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca²⁺]_i by EGCG may be relevant in breast cancer treatment.     

Cav3.1 (alpha1G) controls von Willebrand factor secretion in rat pulmonary microvascular endothelial cells

The T-type Ca2+ channel Cav3.1 subunit is present in pulmonary microvascular endothelial cells (PMVECs), but not in pulmonary artery endothelial cells (PAECs). The present study sought to assess the role of Cav3.1 in thrombin-induced Weibel-Palade body exocytosis and consequent von Willebrand factor (VWF) release. In PMVECs and PAECs transduced with a green fluorescent protein (GFP)-tagged VWF chimera, we examined the real-time dynamics and secretory process of VWF-GFP-containing vesicles in response to thrombin and the cAMP-elevating agent isoproterenol. Whereas thrombin stimulated a progressive decrease in the number of VWF-GFP-containing vesicles in both cell types, isoproterenol only decreased the number of VWF-GFP-containing vesicles in PAECs. In PMVECs, thrombin-induced decrease in the number of VWF-GFP-containing vesicles was nearly abolished by the T-type Ca2+ channel blocker mibefradil as well as by Cav3.1 gene silencing with small hairpin RNA. Expression of recombinant Cav3.1 subunit in PAECs resulted in pronounced increase in thrombin-stimulated Ca2+ entry, which is sensitive to mibefradil. Together, these data indicate that VWF secretion from lung endothelial cells is regulated by two distinct pathways involving Ca2+ or cAMP, and support the hypothesis that activation of Cav3.1 T-type Ca2+ channels in PMVECs provides a unique cytosolic Ca2+ source important for Gq-linked agonist-induced VWF release.     

Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle

Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.     

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.     

Expression and Regulation of Cav3.2 T-Type Calcium Channels during Inflammatory Hyperalgesia in Mouse Dorsal Root Ganglion Neurons

The Cav3.2 isoform of the T-type calcium channel is expressed in primary sensory neurons of the dorsal root ganglion (DRG), and these channels contribute to nociceptive and neuropathic pain in rats. However, there are conflicting reports on the roles of these channels in pain processing in rats and mice. In addition, the function of T-type channels in persistent inflammatory hyperalgesia is poorly understood. We performed behavioral and comprehensive histochemical analyses to characterize Cav3.2-expressing DRG neurons and examined the regulation of T-type channels in DRGs from C57BL/6 mice with carrageenan-induced inflammatory hyperalgesia. We show that approximately 20% of mouse DRG neurons express Cav3.2 mRNA and protein. The size of the majority of Cav3.2-positive DRG neurons (69 ± 8%) ranged from 300 to 700 μm2 in cross-sectional area and 20 to 30 μm in estimated diameter. These channels co-localized with either neurofilament-H (NF-H) or peripherin. The peripherin-positive cells also overlapped with neurons that were positive for isolectin B4 (IB4) and calcitonin gene-related peptide (CGRP) but were distinct from transient receptor potential vanilloid 1 (TRPV1)-positive neurons during normal mouse states. In mice with carrageenan-induced inflammatory hyperalgesia, Cav3.2 channels, but not Cav3.1 or Cav3.3 channels, were upregulated in ipsilateral DRG neurons during the sub-acute phase. The increased Cav3.2 expression partially resulted from an increased number of Cav3.2-immunoreactive neurons; this increase in number was particularly significant for TRPV1-positive neurons. Finally, preceding and periodic intraplantar treatment with the T-type calcium channel blockers mibefradil and NNC 55-0396 markedly reduced and reversed mechanical hyperalgesia during the acute and sub-acute phases, respectively, in mice. These data suggest that Cav3.2 T-type channels participate in the development of inflammatory hyperalgesia, and this channel might play an even greater role in the sub-acute phase of inflammatory pain due to increased co-localization with TRPV1 receptors compared with that in the normal state.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Distinct contributions of different structural regions to the current kinetics of the Cav3.3 T-type Ca2+ channel

Electrophysiological characterization of T-type Ca2+ channel isoforms (Cav3.1, Cav3.2, and Cav3.3) has shown that all of the isoforms are low voltage-activated around resting membrane potential, but their current kinetics are distinctly different, with the activation and inactivation kinetics of the Cav3.1 and Cav3.2 channels being much faster than those of the Cav3.3 channel. We previously reported that multiple structural regions of the Cav3.3 T-type channel participate in determining its current kinetics. Here we have evaluated the relative contributions of individual cytoplasmic and trans-membrane regions to the current kinetics of the channel, by systematically replacing individual regions of Cav3.3 with the corresponding regions of Cav3.1. Introduction of the Cav3.1 III-IV loop into the Cav3.3 backbone accelerated both the activation and inactivation kinetics more prominently than any other intracellular loop or tail. Among the trans-membrane domains, introduction of the domain I of Cav3.1 into Cav3.3 accelerated both the activation and inactivation kinetics most effectively. These findings suggest that the current kinetics of the Cav3.3 channel are differentially controlled by several structural regions, among which the III-IV loop and domain I are the most prominent in governing both activation and inactivation kinetics.     

Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop

Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (Δ1-Cav3.2; Ser⁴²³–Pro⁵⁴²) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, Δ1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of Δ1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels.     

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

Low voltage-activated (LVA) T-type Ca²⁺ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca²⁺ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Ca_v3.1 to Ca_v3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca²⁺ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Ca_v3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Ca_v3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Ca_v3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca²⁺ channels.     

Chronic social defeat stress-induced enhancement of T-type calcium channels increases burst-firing neurons in the ventral subiculum

The ventral subiculum (vSub), a representative output structure of the hippocampus, serves as a main limbic region in mediating the brain's response to stress. There are three subtypes of subicular pyramidal neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons, located differently along proximal–distal axis. Here, we found that chronic social defeat stress (CSDS) in mice increased the population of SB neurons but decreased RS neurons in the proximal vSub. Specific blockers of T-type calcium channels inhibited the burst firings with a concomitant reduction of afterdepolarization, suggesting that T-type calcium channels underlie the burst-spiking activity. Consistently, CSDS increased both T-type calcium currents and expression of Cav3.1 proteins, a subtype of T-type calcium channels, in the proximal vSub. Therefore, we conclude that CSDS-induced enhancement of Cav3.1 expression increased bursting neuronal population in the vSub, which may contribute to stress-related behaviors.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca²⁺ is essential for cardiomyocyte function, and alterations in proteins that regulate Ca²⁺ influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca²⁺ channels are one pathway of Ca²⁺ entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Ca_v3.1 (α1G) and Ca_v3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca²⁺ channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Ca_v3.1 and Ca_v3.2 Ca²⁺ channel expression in the pathological setting. The putative association of T-type Ca²⁺ channels with Ca²⁺ dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Gating of the expressed Cav3.1 calcium channel

Intramembrane charge movement originating from Cav3.1 (T-type) channel expressed in HEK 293 cells was investigated. Ion current was blocked by 1 mM La3+. Charge movement was detectable for depolarizations above approximately -70 mV and saturated above +60 mV. The voltage dependence of charge movement followed a single Boltzmann function with half-maximal activation voltage +12.9 mV and +12.3 mV and with slopes of 22.4 mV and 18.1 mV for the ON- and OFF-charge movement, respectively. Inactivation of I(Ca) by prolonged depolarization pulse did not immobilize intramembrane charge movement in the Cav3.1 channel.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.     

Molecular characterization of T-type calcium channels

Molecular cloning of the low voltage-gated, T-type, calcium channel family opened new avenues of research into their structure-function, distribution, pharmacology, and regulation. Cloning of mammalian cDNAs led to the identification of three T-channel genes: CACNA1G, encoding Cav3.1; CACNA1H, encoding Cav3.2; and CACNA1I, encoding Cav3.3. This allowed sequencing of these genes in absence epilepsy patients, and the identification of single nucleotide polymorphisms (SNPs) that alter channel activity. Their distribution in thalamic nuclei, coupled with the physiological role they play in thalamic oscillations, leads to the conclusion that SNPs in T-channel genes may contribute to neurological disorders characterized by thalamocortical dysrhythmia, such as generalized epilepsy. This section reviews the structure of T-channels, how splicing affects structure and function, how SNPs alter channel activity, and how high voltage-activated auxiliary subunits affect T-channels.