Regulation and function of Cav3.1 T-type calcium channels in IGF-I-stimulated pulmonary artery smooth muscle cells

Arterial smooth muscle cells enter the cell cycle and proliferate in conditions of disease and injury, leading to adverse vessel remodeling. In the pulmonary vasculature, diverse stimuli cause proliferation of pulmonary artery smooth muscle cells (PASMCs), pulmonary artery remodeling, and the clinical condition of pulmonary hypertension associated with significant health consequences. PASMC proliferation requires extracellular Ca(2+) influx that is intimately linked with intracellular Ca(2+) homeostasis. Among the primary sources of Ca(2+) influx in PASMCs is the low-voltage-activated family of T-type Ca(2+) channels; however, up to now, mechanisms for the action of T-type channels in vascular smooth muscle cell proliferation have not been addressed. The Ca(v)3.1 T-type Ca(2+) channel mRNA is upregulated in cultured PASMCs stimulated to proliferate with insulin-like growth factor-I (IGF-I), and this upregulation depends on phosphatidylinositol 3-kinase/Akt signaling. Multiple stimuli that trigger an acute rise in intracellular Ca(2+) in PASMCs, including IGF-I, also require the expression of Ca(v)3.1 Ca(2+) channels for their action. IGF-I also led to cell cycle initiation and proliferation of PASMCs, and, when expression of the Ca(v)3.1 Ca(2+) channel was knocked down by RNA interference, so were the expression and activation of cyclin D, which are necessary steps for cell cycle progression. These results confirm the importance of T-type Ca(2+) channels in proper progression of the cell cycle in PASMCs stimulated to proliferate by IGF-I and suggest that Ca(2+) entry through Ca(v)3.1 T-type channels in particular interacts with Ca(2+)-dependent steps of the mitogenic signaling cascade as a central component of vascular remodeling in disease.     

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+). Biophysical and pharmacological data indicated that the Ca(2+) current is predominantly mediated by T-type (Ca(v)3.2) channels. The number of cells expressing T-type channels and Ca(v)3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca(2+) currents with Ni(2+) induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Ca(v)3.2) Ca(2+) channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Ca(v)3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca(2+) entry mediated by Ca(v)3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.     

L-type calcium channels in growth plate chondrocytes participate in endochondral ossification

Longitudinal bone growth occurs by a process called endochondral ossification that includes chondrocyte proliferation, differentiation, and apoptosis. Recent studies have suggested a regulatory role for intracellular Ca(2+) (Ca(i) (2+)) in this process. Indirect studies, using Ca(2+) channel blockers and measurement of Ca(i) (2+), have provided evidence for the existence of Ca(2+) channels in growth plate chondrocytes. Furthermore, voltage-gated Ca(2+) channels (VGCC), and specifically L- and T-type VGCCs, have been recently described in murine embryonic growth plates. Our aim was to assess the effect of L-type Ca(2+) channel blockers on endochondral ossification in an organ culture. We used cultures of fetal rat metatarsal rudiments at 20 days post gestational age, with the addition of the L-type Ca(2+) channel blockers verapamil (10-100 microM) or diltiazem (10-200 microM) to the culture medium. Longitudinal bone growth, chondrocyte differentiation (number of hypertrophic chondrocytes), and cell proliferation (incorporation of tritiated thymidine) were measured. Verapamil dose-dependently decreased growth, the number of hypertrophic chondrocytes, and cell proliferation, at concentrations of 10-100 microM. Growth and the number of hypertrophic chondrocytes decreased significantly with diltiazem at 50-100 microM, and proliferation decreased significantly at concentrations of 10-200 microM. Additionally, there was no increase in apoptosis over physiological levels with either drug. We confirmed the presence of L-type VGCCs in rat rudiments using immunohistochemistry, and showed that the antagonists did not alter the pattern of VGCC expression. In conclusion, our data suggest that L-type Ca(2+) channel activity in growth plate chondrocytes is necessary for normal longitudinal growth, participating in chondrocyte proliferation and differentiation.     

Selectivity signatures of three isoforms of recombinant T-type Ca2+ channels

Voltage-gated Ca(2+) channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca(2+) over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca(2+) permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca(2+)-channel alpha1 subunits, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca(v)3 channels select Ca(2+) over Na(+) by affinity. Ca(v)3.1 and Ca(v)3.2 discriminate Ca(2+), Sr(2+) and Ba(2+) based on the ion's effects on the open channel probability, whilst Ca(v)3.3 discriminates based on the ion's intrapore binding affinity. All Ca(v)3s were characterized by much smaller difference in the K(D) values for Na(+) current blockade by Ca(2+) (K(D1) approximately 6 microM) and for Ca(2+) current saturation (K(D2) approximately 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na(+)/Ca(2+) current at close to physiological Ca(2+) concentrations, which was the strongest for Ca(v)3.3, smaller for Ca(v)3.2 and the smallest for Ca(v)3.1. In addition to intrapore Ca(2+) binding site(s) Ca(v)3.2, but not Ca(v)3.1 and Ca(v)3.3, is likely to possess an extracellular Ca(2+) binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca(2+) current in native cells.     

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.     

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Loss of the cellular prion protein affects the Ca2+ homeostasis in hippocampal CA1 neurons

Previous neurophysiological studies on prion protein deficient (Prnp(-/-)) mice have revealed a significant reduction of slow afterhyperpolarization currents (sI(AHP)) in hippocampal CA1 pyramidal cells. Here we aim to determine whether loss of PrP(C.) directly affects the potassium channels underlying sI(AHP) or if sI(AHP) is indirectly disturbed by altered intracellular Ca(2+) fluxes. Patch-clamp measurements and confocal Ca(2+) imaging in acute hippocampal slice preparations of Prnp(-/-) mice compared to littermate control mice revealed a reduced Ca(2+) rise in CA1 neurons lacking PrP(C) following a depolarization protocol known to induce sI(AHP). Moreover, we observed a reduced Ca(2+) influx via l-type voltage gated calcium channels (VGCCs). No differences were observed in the protein expression of the pore forming alpha1 subunit of VGCCs Prnp(-/-) mice. Surprisingly, the beta2 subunit, critically involved in the transport of the alpha1 subunit to the plasma membrane, was found to be up-regulated in knock out hippocampal tissue. On mRNA level however, no differences could be detected for the alpha1C, D and beta2-4 subunits. In conclusion our data support the notion that lack of PrP(C.) does not directly affect the potassium channels underlying sI(AHP), but modulates these channels due to its effect on the intracellular free Ca(2+) concentration via a reduced Ca(2+) influx through l-type VGCCs.     

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.     

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.     

Extrinsic regulation of T-type Ca(2+) channel expression in chick nodose ganglion neurons

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.     

Ghrelin inhibits proliferation and increases T-type Ca2+ channel expression in PC-3 human prostate carcinoma cells

Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca(2+) levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca(2+) channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca(2+) channel expression.     

Modulation of native T-type calcium channels by omega-3 fatty acids

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.     

Epigenetic reprogramming as a key contributor to melanocyte malignant transformation

Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

Two populations of pancreatic islet alpha-cells displaying distinct Ca2+ channel properties

In low or absence of glucose, alpha-cells generate rhythmic action potentials and secrete glucagon. alpha-Cell T-type Ca(2+) channels are believed to be pacemaker channels, which are expected to open near the resting membrane potential (around -60 mV) to initiate a small depolarization. A previous publication, however, showed that alpha-cell T-type Ca(2+) channels have an activation threshold of -40 mV, which does not appear to fulfill their role as pacemakers. In this work, we investigated the Ca(2+) channel characteristics in alpha-cells of mouse-insulin-promoter green-fluorescent-protein (MIP-GFP) mouse. The beta-cells of MIP-GFP were conveniently distinguished as green cells, while immunostaining indicated that the majority of non-green cells were alpha-cells. We found that majority of alpha-cells possessed T-type Ca(2+) channels having an activation threshold of -40 mV; these cells also had high-voltage-activated (HVA) Ca(2+) channels (activation threshold of -20 mV). A novel finding here is that a minority of alpha-cells had T-type Ca(2+) channels with an activation threshold of -60 mV. This minor population of alpha-cells was, surprisingly, devoid of HVA Ca(2+) channels. We suggest that this alpha-cell subpopulation may act as pacemaker cells in low or absence of glucose.     

T‐type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T‐type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3‐II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).     

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.     

ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.