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 CaV3 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.
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, CaV3.1 (α1G), CaV3.2 (α1H), and CaV3.3 (α1I), which functionally can be distinguished by their electrophysiological properties1,5. CaV3.1 and CaV3.2 are the most commonly expressed subunits generating T-type Ca2+ current (ICa,T) in brain and heart. CaV3.2 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 CaV 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 CaV3.1 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 CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The increase in CaV3.1 protein levels demonstrated by western blot analysis of cultured murine cardiac cells (HL-1) using Anti-CACNA1G (CaV3.1) 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 ICa,T through the activation of Src protein kinase. Immunoprecipitation of mouse HL-1 cells or mouse heart lysates using Anti-CACNA1G (CaV3.1) Antibody showed that Src immunoprecipitated with CaV3.1. In addition, CaV3.1 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 CaV3.1 and CaV3.3 channel subunits in reticular thalamic nucleus (RE) of the cat. Immunohistochemical staining using Anti-CACNA1G (CaV3.1) Antibody and Anti-CaV3.3 (CACNA1I) Antibody (#ACC-009) demonstrated that CaV3.1 is predominantly localized to the somata and proximal dendrites and CaV3.3 channels in cell bodies. CaV3.3 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 CaV3.1 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 CaV3.3 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 CaV3.1 for which its expression was verified by western blot using Anti-CACNA1G (CaV3.1) Antibody.
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 CaV3.1 and CaV3.2 channels as shown in western blot and immunocytochemistry using Anti-CACNA1G (CaV3.1) Antibody and Anti-CaV3.2 (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 CaV3.1 and CaV3.2 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 CaV3.1. In addition, immunohistochemical staining of mouse carotid bodies using Anti-CACNA1G (CaV3.1) Antibody showed that the channel was upregulated in VSMCs during neointimal formation in injured carotid arteries (Figure 1). Under control conditions and in CaV3.1 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 CaV3.1 is required for VSMC proliferation during neointimal formation.
Figure 1. Expression of CaV3.1 in Mouse CarotidImmunohistochemical staining of mouse carotid sections using Anti-CACNA1G (CaV3.1) Antibody (#ACC-021). CaV3.1 expression (upper panel), (green) is upregulated following injury and not detected in wildtype and CaV3.1-/- mouse.Adapted from reference 20 with permission of the European Society of Cardiology.
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 CaV3.2 channel colocalized with syntaxin-1A in immunocytochemical staining of rat nRT neurons (Figure 2). This interaction modulates CaV3.2 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 CaV3.2 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 CaV3.2 T-type Ca2+ channels contribute to low-threshold exocytosis.
Figure 2. Syntaxin-1A Co-Immunoprecipitates and Immuno-Colocalizes with CaV3.2A. Syntaxin-1A is co-immunoprecipitated from rat brain with T-type channels using Anti-CACNA1G (CaV3.1) Antibody (#ACC-021), Anti-CaV3.2 (CACNA1H) Antibody (#ACC-025) and Anti-CaV3.3 (CACNA1I) Antibody (#ACC-009). B. Immunocytochemical staining of rat reticular thalamic neurons using Anti-CaV3.2 (CACNA1H) Antibody (upper left panel). CaV3.2 colocalizes with Syntaxin-1A (lower left panel).Adapted from reference 21 with permission of The American Society for Biochemistry and Molecular Biology.
Release of conventional neurotransmitters is mainly controlled by Ca2+ influx via high-voltage-activated (HVA) CaV channels but there is little evidence that low-voltage-activated CaV 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 CaV3.1 and Ca2+ from internal stores. Anti-CACNA1G (CaV3.1) 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).
Figure 3. Expression of CaV3.1 in Rat HippocampusA. Immunohistochemical staining of rat hippocampus using Anti-CACNA1G (CaV3.1) Antibody (#ACC-021). CaV3.1 expression in CA1 region is blocked when preincubating with the negative control antigen (inset). B. Immunofluorescent labeling of the same sections shows that CaV3.1 co-localizes with Tau1, a marker for neuronal axons (lower panel).Adapted from reference 17 with permission of the Society for Neuroscience.
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-CaV3.2 (CACNA1H) Antibody showed that the channel is expressed. Function of the channel is necessary for catecholamine release in response to acute hypoxia. CaV3.2 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 CaV3.2 calcium influx in mouse chromaffin cells of the adrenal medulla Anti-CaV3.2 (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 CaV3.2 Ca2+ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.
Figure 4. Expression of CaV3.2 in Mouse Adrenal GlandsImmunohistochemical staining of mouse adrenal gland sections using Anti-CaV3.2 (CACNA1H) Antibody (#ACC-025). CaV3.2 co-localizes with thyrosine hydroxylase (TH), a marker for cathecholaminesecreting adrenal chromaffin cells (third panel). PACAP expression (purple) is detected in the surrounding area of CaV3.2 expressing cells (right panel).Adapted from reference 2 with permission of The American Society for Biochemistry and Molecular Biology.
Excitation-contraction coupling in afferent arterioles requires the activation of CaV 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, CaV3.1 was responsible for the K+-elicited constriction which was inhibited with a T-type blocker. CaV3.2 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-CaV3.2 (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.
Alomone Labs TTA-P2 inhibits T-type CaV channels heterologously expressed in Xenopus oocytesA. Time course of CaV3.2 peak current amplitude, elicited by 100 ms voltage step from holding potential of -100mV to -30 mV, delivered every 10 seconds. Application of 5 μM TTA-P2 (#T-155) inhibits the CaV3.2 current in a reversible manner (indicated by the horizontal bar). B. Representative current traces before and during application of 5 μM TTA-P2 as indicated.
1. Chemin, J. et al. (2002) J. Physiol. 540, 3.
2. Hill, J. et al. (2011) J. Biol. Chem. 286, 42459.
3. Huc, S. et al. (2009) Biochim. Biophys. Acta 1793, 947.
4. Kovacs, K. et al. (2010) J. Neurosci. Res. 88, 448.
5. Kozlov, A.S. et al. (1999) Eur. J. Neurosci. 11, 4149.
6. Kunzelmann, K.J. (2005) Membr. Biol. 205, 159.
7. Levitsky, K.L. and Lopez-Barneo, J. (2009) J. Physiol. 587, 1917.
8. McGivern, J.G. (2006) Drug Discov. Today 11, 245.
9. McKay, B.E. et al. (2006) Eur. J. Neurosci. 24, 2581.
10. Mor, M. et al. (2012) Am. J. Physiol. 303, C192.
11. Moriguchi, S. et al. (2012) J. Neurochem. 121, 44.
12. Neher, E. and Sakaba, T. (2008) Neuron 59, 861.
13. Pan, Z.H. et al. (2001) Neuron 32, 89.
14. Poulsen, C.B. et al. (2011) Kidney Int. 79, 443.
15. Rao, F. et al. (2013) Exp. Physiol. 98, 172.
16. Segal, D. et al. (2004) Biochem. Biophys. Res. Commun. 323, 1145.
17. Tang, A.H. et al. (2011) J. Neurosci. 31, 13546.
18. Taylor, J.T. et al. (2008) Cancer Lett. 267, 116.
19. Toledo, A. et al. (2012) Cell Calcium 52, 377.
20. Tzeng, B.H. et al. (2012) Cardiovasc. Res. 96, 533.
21. Weiss, N. et al. (2012) J. Biol. Chem. 287, 2810.