T-Type Ca²⁺ Channels

T-type Ca²⁺ channels are at the heart of numerous research topics such as pain and epilepsy. This paper aims at describing various aspects of Ca_V3 regulation, their role in LTP, cell proliferation, exocytosis and neurotransmitter release and finally, their role in excitation-contraction in afferent arterioles using Alomone Labs products.

Introduction

T-type Ca²⁺ channels (TTCC) are low VGCCs, expressed in various tissues including brain and heart and contribute to a variety of physiological functions such as neuronal excitability, hormone secretion, muscle contraction, and pacemaker activity³. There is an increasing body of evidence that TTCC can trigger fast and low-threshold exocytosis¹³.

Molecular cloning studies have identified three different TTCC isoforms, Ca_V3.1 (α_1G), Ca_V3.2 (α_1H), and Ca_V3.3 (α_1I), which functionally can be distinguished by their electrophysiological properties^1,5. Ca_V3.1 and Ca_V3.2 are the most commonly expressed subunits generating T-type Ca²⁺ current (I_Ca,T) in brain and heart. Ca_V3.2 T-type Ca²⁺ channels are involved in neurological disorders such as epilepsy and pain⁸.

Regulation of T-Type Channels

Growth factors and hormones have regulatory effects on the functional expression of Ca_V channels. Toledo et al.¹⁹ investigated whether the actions of insulin on T-type channel functional expression are mediated by transcriptional and/or post-transcriptional mechanisms. Results indicate that chronic treatment of rat pituitary-derived GH3 cells with insulin may regulate the incorporation and recycling of surface membrane of Ca_V3.1 channels in HEK-293 cells. Thus, effects of insulin may require post-transcriptional regulation. The expression of all three T-type channels was verified by immunocytochemical staining of GH3 cells using the respective Alomone Labs antibodies.

Zinc transporter-1 (ZnT-1), a putative zinc transporter that confers cellular resistance from zinc toxicity, has important regulatory functions, including inhibition of L-type Ca²⁺ channels and activation of Raf-1 kinase¹⁶. Mor et al.¹⁰ found that ZnT-1 enhances the activity of Ca_V3.1 and Ca_V3.2 through activation of Ras-ERK signaling. The increase in Ca_V3.1 protein levels demonstrated by western blot analysis of cultured murine cardiac cells (HL-1) using Anti-CACNA1G (Ca_V3.1) Antibody (#ACC-021) is associated with enhanced trafficking of the channel to the plasma membrane.

The role of macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, in the regulation of T-type Ca²⁺ channels in atrial myocytes was recently investigated¹⁵. Expression levels of TTCC mRNA were significantly reduced in patients with atrial fibrillation (AF) and MIF suppressed I_Ca,T through the activation of Src protein kinase. Immunoprecipitation of mouse HL-1 cells or mouse heart lysates using Anti-CACNA1G (Ca_V3.1) Antibody showed that Src immunoprecipitated with Ca_V3.1. In addition, Ca_V3.1 and Src co-localize in immunocytochemical staining of HL-1 cells. Overall, the data show that MIF is involved in the pathology of AF by decreasing the T-type Ca²⁺ current in atrium-derived myocytes through impairment of channel function.

Burst Firing and LTP

Low-voltage-activated Ca²⁺ channels play a critical role in the generation of burst firing in the thalamus. Kovacs et al.⁴ investigated the subcellular expression pattern of Ca_V3.1 and Ca_V3.3 channel subunits in reticular thalamic nucleus (RE) of the cat. Immunohistochemical staining using Anti-CACNA1G (Ca_V3.1) Antibody and Anti-Ca_V3.3 (CACNA1I) Antibody (#ACC-009) demonstrated that Ca_V3.1 is predominantly localized to the somata and proximal dendrites and Ca_V3.3 channels in cell bodies. Ca_V3.3 isoform was absent from large caliber, presumably proximal dendritic segments. These results suggest that compartmentalization of distinct T-type channel subunits occurs along the dendrites of RE cells and correlates with observations that Ca_V3.1 isoforms are expressed in the proximal region of many cell types. In contrast, synaptic inputs arriving at distal dendrites are more likely to be modified by the Ca_V3.3 subunit, which is frequently localized in thin dendritic segments⁹. These data emphasize the existence of multiple T-type channel variants with uneven, predominantly dendritic localization in RE cells. Immunogold staining of the channels showed similar results.

A connection between T-type VGCCs and the enhancement of cortical long term potentiation (LTP) was established¹¹. ST101 improved cognition in the CNS by activating T-type channels. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation. Notably, enhanced CaMKII autophosphorylation following ST101 treatment significantly decreased when pre-treating with a T-type VGCC inhibitor. Similarly, enhanced LTP in cortical slices was completely inhibited by T-type blocker treatment. In addition, ST101 directly enhanced T-type VGCC currents in Neuro2A (N2A) neuroblastoma cells over-expressing recombinant human Ca_V3.1 for which its expression was verified by western blot using Anti-CACNA1G (Ca_V3.1) Antibody.

Cell Proliferation

Several recent reviews have implied that Ca²⁺ channels play a role in regulating Ca²⁺ signaling during cell proliferation⁶. A study showed that T-type Ca²⁺ channels are predominately expressed in the fast growing phase of the breast cancer cells MCF-7 (ERα+), suggesting that they play a role in cancer cell proliferation¹⁸. MCF-7 cells express both Ca_V3.1 and Ca_V3.2 channels as shown in western blot and immunocytochemistry using Anti-CACNA1G (Ca_V3.1) Antibody and Anti-Ca_V3.2 (CACNA1H) Antibody (#ACC-025). Treating MCF-7 cells with a specific T-type blocker inhibited cell proliferation. In contrast, treating MCF-10A cells, a noncancer breast epithelial cell line, had no effect. In addition siRNA treatment targeting both Ca_V3.1 and Ca_V3.2 resulted in significantly lower proliferation rates in MCF-7 cells compared to control. These results suggest that T-type Ca²⁺ channel inhibition may reduce cellular proliferation in mitogenic breast cancer cells.

Another study examined the role of low-voltage gated T-type Ca²⁺ channels in regulating vascular smooth muscle cell (VSMC) proliferation²⁰ during the narrowing of blood vessels, caused by neointimal formation. Injury-induced neointimal formation was abolished in mice lacking Ca_V3.1. In addition, immunohistochemical staining of mouse carotid bodies using Anti-CACNA1G (Ca_V3.1) Antibody showed that the channel was upregulated in VSMCs during neointimal formation in injured carotid arteries (Figure 1). Under control conditions and in Ca_V3.1 knockout mice, the channel was undetectable. It was also found that a T-type channel blocker was able to reduce the neointimal formation after vascular injury in wild-type mice. These findings demonstrate that Ca_V3.1 is required for VSMC proliferation during neointimal formation.

Exocytosis and Neurotransmitter Release

A study was initiated in order to gain insights regarding the regulation of T-type Ca²⁺ channels by SNARE proteins in low-threshold exocytosis²¹. Although syntaxin-1A co-immunoprecipitated with all three T-type channels using Alomone Labs T-type channel antibodies, only Ca_V3.2 channel colocalized with syntaxin-1A in immunocytochemical staining of rat nRT neurons (Figure 2). This interaction modulates Ca_V3.2 channel gating and regulates T-type channel-mediated exocytosis. Syntaxin-1A binding to a synaptic protein interaction site (synprint) located within the intracellular II-III linker region of the channel and the disruption of Ca²⁺ channel-SNARE coupling alters neurotransmitter release¹⁴. Electrophysiological analysis of Ca_V3.2 channels in HEK-293 cells (tsA-201 cells) revealed that syntaxin-1A potently decreases channel availability by shifting the steady-state inactivation toward more hyperpolarized potentials. These data provide a molecular mechanism by which Ca_V3.2 T-type Ca²⁺ channels contribute to low-threshold exocytosis.

Release of conventional neurotransmitters is mainly controlled by Ca²⁺ influx via high-voltage-activated (HVA) Ca_V channels but there is little evidence that low-voltage-activated Ca_V channels (T-type) also take part¹². A study investigated whether T-type Ca²⁺ channels are involved in regulating GABA transmission from cortical perisomatic targeting interneurons in rat hippocampal CA1¹⁷. Results indicate that activation of axonal nicotinic acetylcholine receptors (nAChRs) can stimulate GABA release via a mechanism that depends on Ca_V3.1 and Ca²⁺ from internal stores. Anti-CACNA1G (Ca_V3.1) Antibody was used to show the expression of the channel in immunohistochemical staining of rat hippocampal slices, which was blocked when incubated with the negative control antigen (Figure 3A). Immunofluorescent studies show that the channel co-localizes with Tau1, a marker of neuronal axons (Figure 3B).

Neonatal chromaffin cells of the adrenal medulla (AM) are intrinsic chemoreceptors that secrete catecholamines in response to hypoxia. A study investigated the contribution of T-type Ca²⁺ channels to the developmental changes in the chemoreceptive properties of these cells⁷. Immunohistochemical staining of rat adrenal glands and immunocytochemical staining of rat chromaffin cells, both with Anti-Ca_V3.2 (CACNA1H) Antibody showed that the channel is expressed. Function of the channel is necessary for catecholamine release in response to acute hypoxia. Ca_V3.2 expression and chromaffin cell response to hypoxia decrease with postnatal maturation, indicating that T-type Ca²⁺ channels are essential for the acute response of chromaffin cells to hypoxia.

In a similar work, Hill et al. ² examined the effects of acute sympathetic stress on T-type Ca_V3.2 calcium influx in mouse chromaffin cells of the adrenal medulla Anti-Ca_V3.2 (CACNA1H) Antibody was used to detect the channel in these cells (Figure 4). Pituitary adenylate cyclase activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. It was found that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phospho-dependent recruitment of Ca_V3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.

Contraction

Excitation-contraction coupling in afferent arterioles requires the activation of Ca_V channels. Poulsen et al.¹⁴ investigated the role of T-type channels in the regulation of cortical efferent arteriolar tone. Depolarization of mouse perfused cortical efferent arterioles consistently constricted the blood vessels. Furthermore, Ca_V3.1 was responsible for the K⁺-elicited constriction which was inhibited with a T-type blocker. Ca_V3.2 was found to be involved in the spontaneous dilatation that occurs in the blood vessels after the initial constriction. Immunohistochemical staining of rat kidney sections using Anti-Ca_V3.2 (CACNA1H) Antibody demonstrated that the channel is present in arteries and arterioles. These data suggest that T-type voltage-gated Ca²⁺ channels are functionally important for depolarization induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.

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