Inhibition Of T-type Channels Research Articles

T- and L-Type Calcium Channels Maintain Calcium Oscillations in the Murine Zona Glomerulosa.

The zona glomerulosa of the adrenal gland is responsible for the synthesis and release of the mineralocorticoid aldosterone. This steroid hormone regulates salt reabsorption in the kidney and blood pressure. The most important stimuli of aldosterone synthesis are the serum concentrations of angiotensin II and potassium. In response to these stimuli, voltage and intracellular calcium levels in the zona glomerulosa oscillate, providing the signal for aldosterone synthesis. It was proposed that the voltage-gated T-type calcium channel CaV3.2 is necessary for the generation of these oscillations. However, Cacna1h knock-out mice have normal plasma aldosterone levels, suggesting additional calcium entry pathways. We used a combination of calcium imaging, patch clamp, and RNA sequencing to investigate calcium influx pathways in the murine zona glomerulosa. Cacna1h-/- glomerulosa cells still showed calcium oscillations with similar concentrations as wild-type mice. No calcium channels or transporters were upregulated to compensate for the loss of CaV3.2. The calcium oscillations observed were instead dependent on L-type voltage-gated calcium channels. Furthermore, we found that L-type channels can also partially compensate for an acute inhibition of CaV3.2 in wild-type mice. Only inhibition of both T- and L-type calcium channels abolished the increase of intracellular calcium caused by angiotensin II in wild-type. Our study demonstrates that T-type calcium channels are not strictly required to maintain glomerulosa calcium oscillations and aldosterone production. Pharmacological inhibition of T-type channels alone will likely not significantly impact aldosterone production in the long term.

MEPIRAPIM-derived synthetic cannabinoids inhibit T-type calcium channels with divergent effects on seizures in rodent models of epilepsy.

Background: T-type Ca2+ channels (Cav3) represent emerging therapeutic targets for a range of neurological disorders, including epilepsy and pain. To aid the development and optimisation of new therapeutics, there is a need to identify novel chemical entities which act at these ion channels. A number of synthetic cannabinoid receptor agonists (SCRAs) have been found to exhibit activity at T-type channels, suggesting that cannabinoids may provide convenient chemical scaffolds on which to design novel Cav3 inhibitors. However, activity at cannabinoid type 1 (CB1) receptors can be problematic because of central and peripheral toxicities associated with potent SCRAs. The putative SCRA MEPIRAPIM and its analogues were recently identified as Cav3 inhibitors with only minimal activity at CB1 receptors, opening the possibility that this scaffold may be exploited to develop novel, selective Cav3 inhibitors. Here we present the pharmacological characterisation of SB2193 and SB2193F, two novel Cav3 inhibitors derived from MEPIRAPIM. Methods: The potency of SB2193 and SB2193F was evaluated in vitro using a fluorometric Ca2+ flux assay and confirmed using whole-cell patch-clamp electrophysiology. In silico docking to the cryo-EM structure of Cav3.1 was also performed to elucidate structural insights into T-type channel inhibition. Next, in vivo pharmacokinetic parameters in mouse brain and plasma were determined using liquid chromatography-mass spectroscopy. Finally, anticonvulsant activity was assayed in established genetic and electrically-induced rodent seizure models. Results: Both MEPIRAPIM derivatives produced potent inhibition of Cav3 channels and were brain penetrant, with SB2193 exhibiting a brain/plasma ratio of 2.7. SB2193 was further examined in mouse seizure models where it acutely protected against 6Hz-induced seizures. However, SB2193 did not reduce spontaneous seizures in the Scn1a +/- mouse model of Dravet syndrome, nor absence seizures in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Surprisingly, SB2193 appeared to increase the incidence and duration of spike-and-wave discharges in GAERS animals over a 4h recording period. Conclusion: These results show that MEPIRAPIM analogues provide novel chemical scaffolds to advance Cav3 inhibitors against certain seizure types.

Role of T CD4+ cells, macrophages, C-low threshold mechanoreceptors and spinal Cav 3.2 channels in inflammation and related pain-like symptoms in murine inflammatory models.

T-type calcium channels, mainly the Cav 3.2 subtype, are important contributors to the nociceptive signalling pathway. We investigated their involvement in inflammation and related pain-like symptoms. The involvement of Cav 3.2 and T-type channels was investigated using genetic and pharmacological inhibition to assess mechanical allodynia/hyperalgesia and oedema development in two murine inflammatory pain models. The location of Cav 3.2 channels involved in pain-like symptoms was studied in mice with Cav 3.2 knocked out in C-low threshold mechanoreceptors (C-LTMR) and the use of ABT-639, a peripherally restricted T-type channel inhibitor. The anti-oedema effect of Cav 3.2 channel inhibition was investigated in chimeric mice with immune cells deleted for Cav 3.2. Lymphocytes and macrophages from either green fluorescent protein-targeted Cav 3.2 or KO mice were used to determine the expression of Cav 3.2 protein and the functional status of the cells. Cav 3.2 channels contributed to the development of pain-like symptoms and oedema in the two murine inflammatory pain models. Our results provided evidence of the involvement of Cav 3.2 channels located on C-LTMRs and spinal cord in inflammatory pain. Cav 3.2 channels located in T cells and macrophages contribute to the inflammatory process. Cav 3.2 channels play crucial roles in inflammation and related pain, implying that targeting of Cav 3.2 channels with pharmacological agents could be an attractive and readily evaluable strategy in clinical trials, to relieve chronic inflammatory pain in patients.

The T-type calcium channel antagonist, Z944, reduces spinal excitability and pain hypersensitivity.

Background and PurposeT‐type voltage‐gated calcium channels are an emerging therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T‐type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T‐type antagonists failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T‐type channels alone may be insufficient for pain relief.Experimental ApproachWe utilized the selective and CNS‐penetrant T‐type channel antagonist, Z944, in electrophysiological, calcium imaging and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours.Key ResultsVoltage‐clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T‐type current, which was blocked by Z944. Due to this highly prevalent T‐type current, Z944 potently blocked action‐potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1–10 mg·kg−1) dose‐dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats.Conclusion and ImplicationsT‐type calcium channels critically shape the excitability of lamina I pain processing neurons and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.

Synthesis and biological evaluation of pyrrolidine-based T-type calcium channel inhibitors for the treatment of neuropathic pain

The treatment of neuropathic pain is one of the urgent unmet medical needs and T-type calcium channels are promising therapeutic targets for neuropathic pain. Several potent T-type channel inhibitors showed promising in vivo efficacy in neuropathic pain animal models and are being investigated in clinical trials. Herein we report development of novel pyrrolidine-based T-type calcium channel inhibitors by pharmacophore mapping and structural hybridisation followed by evaluation of their Cav3.1 and Cav3.2 channel inhibitory activities. Among potent inhibitors against both Cav3.1 and Cav3.2 channels, a promising compound 20n based on in vitro ADME properties displayed satisfactory plasma and brain exposure in rats according to in vivo pharmacokinetic studies. We further demonstrated that 20n effectively improved the symptoms of neuropathic pain in both SNL and STZ neuropathic pain animal models, suggesting modulation of T-type calcium channels can be a promising therapeutic strategy for the treatment of neuropathic pain.

T-type Ca2 + channels elicit pro-proliferative and anti-apoptotic responses through impaired PP2A/Akt1 signaling in PASMCs from patients with pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (iPAH) is characterized by obstructive hyperproliferation and apoptosis resistance of distal pulmonary artery smooth muscle cells (PASMCs). T-type Ca2+ channel blockers have been shown to reduce experimental pulmonary hypertension, although the impact of T-type channel inhibition remains unexplored in PASMCs from iPAH patients. Here we show that T-type channels Cav3.1 and Cav3.2 are present in the lung and PASMCs from iPAH patients and control subjects. The blockade of T-type channels by the specific blocker, TTA-A2, prevents cell cycle progression and PASMCs growth. In iPAH cells, T-type channel signaling fails to activate phosphatase PP2A, leading to an increase in ERK1/2, P38 activation. Moreover, T-type channel signaling is redirected towards the activation of the kinase Akt1, leading to increased expression of the anti-apoptotic protein survivin, and a decrease in the pro-apoptotic mediator FoxO3A. Finally, in iPAH cells, Akt1 is no longer able to regulate caspase 9 activation, whereas T-type channel overexpression reverses PP2A defect in iPAH cells but reinforces the deleterious effects of Akt1 activation. Altogether, these data highlight T-type channel signaling as a strong trigger of the pathological phenotype of PASMCs from iPAH patients (hyper-proliferation/cells survival and apoptosis resistance), suggesting that both T-type channels and PP2A may be promising therapeutic targets for pulmonary hypertension.

CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signaling in Rat Retinal Arterioles.

Although L-type Ca2+ channels are known to play a key role in the myogenic reactivity of retinal arterial vessels, the involvement of other types of voltage-gated Ca2+ channels in this process remains unknown. In the present study we have investigated the contribution of T-type Ca2+ channels to myogenic signaling in arterioles of the rat retinal microcirculation. Confocal immunolabeling of whole-mount preparations was used to investigate the localization of CaV3.1-3 channels in retinal arteriolar smooth muscle cells. T-type currents and the contribution of T-type channels to myogenic signaling were assessed by whole-cell patch-clamp recording and pressure myography of isolated retinal arteriole segments. Strong immunolabeling for CaV3.1 was observed on the plasma membrane of retinal arteriolar smooth muscle cells. In contrast, no expression of CaV3.2 or CaV3.3 could be detected in retinal arterioles, although these channels were present on glial cell end-feet surrounding the vessels and retinal ganglion cells, respectively. TTA-A2-sensitive T-type currents were recorded in retinal arteriolar myocytes with biophysical properties distinct from those of the L-type currents present in these cells. Inhibition of T-type channels using TTA-A2 or ML-218 dilated isolated, myogenically active, retinal arterioles. CaV3.1 T-type Ca2+ channels are functionally expressed on arteriolar smooth muscle cells of retinal arterioles and play an important role in myogenic signaling in these vessels. The work has important implications concerning our understanding of the mechanisms controlling blood flow autoregulation in the retina and its disruption during ocular disease.

Abstract 750: Overcoming resistance to anti-EGFR therapy in non-small cell lung cancer with a T-type Ca2+ channels inhibitor

Abstract This study's aim was to define a novel molecular target and to develop an effective agent capable of overcoming intrinsic and acquired resistance to anti-EGFR therapy. Despite introduction of new therapies, lung cancer is a major cause of cancer death. One of its characteristics is the expression and activation of epidermal growth factor receptor (EGFR). Activating mutations in EGFR are present in approximately 20% of patients with non-small cell lung cancer (NSCLC), mostly in never-smokers. While most patients initially respond to treatment with anti-EGFR therapy, essentially all patients develop acquired drug resistance. T-type calcium channels, are present in lung cancer and support cell proliferation. Importantly, their expression often coincides with abnormal expression or mutations in EGFR. In this study, we investigated the effects of T-type channel inhibition on NSCLC survival and resistance to targeted therapy in vitro. We used a panel of cell lines differing in EGFR mutation status and sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Our results show that T-type channel inhibitors alone arrest cells in the G1/G0 phase of the cell cycle and induce cell death. The treatment reduces activity of both PI3K/AKT/mTOR and JAK2/STAT5 pathways, often upregulated as a mechanism of acquired resistance to TKIs. Importantly, combined treatment with TKI, gefitinib, and T-type channels inhibitor, mibefradil, resulted in synergistic growth inhibition. Our results demonstrate a connection between T-type Ca2+ channels and aberrant EGFR activity in NSCLC, and provide the rationale for future use of two targeted therapies together. Since T-type calcium channel inhibitors are already undergoing clinical trials, our discoveries could be quickly translated into clinical practice. Citation Format: Barbara Dziegielewska, Lloyd S. Gray, Jaroslaw Dziegielewski. Overcoming resistance to anti-EGFR therapy in non-small cell lung cancer with a T-type Ca2+ channels inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2015-750

Abstract 1808: Enhancing the Stupp regimen in glioblastoma cancer cells with a T-type calcium channels inhibitor

Abstract Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor. Although introduction of the Stupp regimen combining radiation (IR) with concomitant and adjuvant temozolomide (TMZ) led to increased patient survival, the cure rate of GBM is disappointing. We postulated that sensitization of cancer cells to therapy can be achieved by targeting intracellular pathways and processes. Recently, T-type Ca2+ channels have been proposed as a molecular target for GBM sensitization to chemo- and radiotherapy (Keir et al., 2013; Sheehan et al., 2013). In the present studies, we investigated the effects of T-type channel inhibition on GBM cells’ responses to the Stupp regimen. GBM cells differing in their resistance to TMZ and IR were treated with theT-type channel antagonist, mibefradil, either concomitantly with TMZ or sequentially (Interlaced TherapyTM). Cell viability and clonogenic survival were determined, demonstrating significant enhancement of the Stupp regimen's anticancer effects with co-treatment with T-type channels inhibitors. Importantly for DNA-targeted therapies, we observed a decrease in cells’ ability to repair DNA damage in the presence of mibefradil. The molecular mechanism of this decrease was investigated by assessing expression levels, localization and phosphorylation status of ATM, DNA-PK, γH2AX, Chk1/2, Kap1, p53, Rad51. These mechanistic studies demonstrated that inhibiting T-type Ca2+ channels in GBM affects DNA damage signaling and repair through reduction of repair protein activation and changes their cellular localization. The observed effects are specific for T-type channels inhibition, since L-type channels inhibitors had no effect. Our study provide a rational molecular mechanism for sensitization of GBM to the Stupp regimen by T-type Ca2+ channel inhibition, which will facilitate current and future clinical trials and potentially lead to new treatment options for this deadly brain tumor. Citation Format: Jaroslaw Dziegielewski, Barbara Dziegielewska, Lloyd S. Gray. Enhancing the Stupp regimen in glioblastoma cancer cells with a T-type calcium channels inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1808. doi:10.1158/1538-7445.AM2015-1808

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

T-type channels in the sino-atrial and atrioventricular pacemaker mechanism

Cardiac automaticity is a fundamental physiological function in vertebrates. Heart rate is under the control of several neurotransmitters and hormones and is permanently adjusted by the autonomic nervous system to match the physiological demand of the organism. Several classes of ion channels and proteins involved in intracellular Ca(2+) handling contribute to pacemaker activity. Voltage-dependent T-type Ca(2+) channels are an integral part of the complex mechanism underlying pacemaking. T-type channels also contribute to impulse conduction in mice and humans. Strikingly, T-type channel isoforms are co-expressed in the cardiac conduction system with other ion channels that play a major role in pacemaking such as f- (HCN4) and L-type Cav1.3 channels. Pharmacologic inhibition of T-type channels reduces the spontaneous activity of isolated pacemaker myocytes of the sino-atrial node, the dominant heart rhythmogenic centre. Target inactivation of T-type Cav3.1 channels abolishes I Ca,T in both sino-atrial and atrioventricular myocytes and reduces the daily heart rate of freely moving mice. Cav3.1 channels contribute also to automaticity of the atrioventricular node and to ventricular escape rhythms, thereby stressing the importance of these channels in automaticity of the whole cardiac conduction system. Accordingly, loss-of-function of Cav3.1 channels contributes to severe form of congenital bradycardia and atrioventricular block in paediatric patients.

NMP-7 inhibits chronic inflammatory and neuropathic pain via block of Cav3.2T-type calcium channels and activation of CB2 receptors.

BackgroundT-type calcium channels and cannabinoid receptors are known to play important roles in chronic pain, making them attractive therapeutic targets. We recently reported on the design, synthesis and analgesic properties of a novel T-type channel inhibitor (NMP-7), which also shows mixed agonist activity on CB1 and CB2 receptors in vitro. Here, we analyzed the analgesic effect of systemically delivered NMP-7 (intraperitoneal (i.p.) or intragstric (i.g.) routes) on mechanical hypersensitivity in inflammatory pain induced by Complete Freund’s Adjuvant (CFA) and neuropathic pain induced by sciatic nerve injury.ResultsNMP-7 delivered by either i.p. or i.g. routes produced dose-dependent inhibition of mechanical hyperalgesia in mouse models of inflammatory and neuropathic pain, without altering spontaneous locomotor activity in the open-field test at the highest active dose. Neither i.p. nor i.g. treatment reduced peripheral inflammation per se, as evaluated by examining paw edema and myeloperoxidase activity. The antinociception produced by NMP-7 in the CFA test was completely abolished in CaV3.2-null mice, confirming CaV3.2 as a key target. The analgesic action of intraperitoneally delivered NMP-7 was not affected by pretreatment of mice with the CB1 antagonist AM281, but was significantly attenuated by pretreatment with the CB2 antagonist AM630, suggesting that CB2 receptors, but not CB1 receptors are involved in the action of NMP-7 in vivo.ConclusionsOverall, our work shows that NMP-7 mediates a significant analgesic effect in a model of persistent inflammatory and chronic neuropathic pain by way of T-type channel modulation and CB2 receptor activation. Thus, this study provides a novel therapeutic avenue for managing chronic pain conditions via mixed CB ligands/T-type channel blockers.

Suppression of T-type Ca2+ channels inhibited human laryngeal squamous cell carcinoma cell proliferation running title: roles of T-type Ca2+ channels in LSCC cell proliferation.

Despite the tight correlation between T-type Ca2+ channels and a great variety of tumors, the roles of alpha1G subunit of T-type Ca2+ channels in laryngeal squamous cell carcinoma (LSCC) have not yet been investigated. In the present study, we examined the expression of alpha1G subunit of T-type Ca2+ channel in human LSCC tissues and cell lines. One human laryngeal squamous cell carcinoma cell line, Hep-2, was also examined for T-type channels using voltage-clamp recordings. Cell proliferation assays were performed in the presence or absence of T-type channel blocker mibefradil and alpha1G subunit sepcific siRNA. The cell cycle was determined by flow cytometry. Our results indicated that the a1G subunit of T-type Ca2+ channel is highly expressed in human laryngeal squamous cell carcinoma tissues and cell lines. alpha1G siRNA significantly down-regulated the protein expression of the alpha1G subunit. Both alpha1G siRNA and mibefradil inhibited Hep-2 cell proliferation and arrested cell cycle progression. Together, these findings suggest a functional role of T-type channels in certain laryngeal carcinomas, and that inhibition of T-type channels reduces cell proliferation via cell cycle arrest, suggesting that the alpha1G subunit of T-type Ca2+ channel may be used as a therapeutic target for treating LSCC.

Abstract A239: T-type calcium channels as a novel molecular target for tumor therapy.

Abstract T-type Ca2+ channels are a group of low voltage-gated calcium channels which seem to be crucial for embryonic or stem cell proliferation and differentiation; however, they could be also aberrantly expressed in several human tumors. Since calcium entry is required for smooth transition through the cell cycle, it has been hypothesized that the inhibition of T-type Ca2+ channels blocks cells in G0/G1 phase of cell cycle, while a release from inhibition leads to increased number of cells entering cell cycle and thus increased susceptibility to conventional antitumor therapy. In this study we investigated the effects of T-type channel inactivation, or downregulation, on cell cycle progression, cell death and survival, and resistance to radiotherapy (RT). The experiments were conducted using several cancer cell lines expressing T-type Ca2+ channels (glioblastoma, colon and prostate) and selective and structurally unrelated calcium channels antagonists, including Mibefradil, a drug that is currently undergoing clinical trial for its antitumor properties, and is planned to be tested as radiosensitizing agent in recurrent gliobastoma tumors. The effects of T-type Ca2+ channel inhibition on cell cycle progression, cell death and the expression/activation of cell cycle regulated proteins were assessed. Finally, the combination treatments including T-type channel antagonist and radiotherapy were assayed. We show that T-type Ca2+ channels are important for cell cycle progression and resistance to RT, and that their inhibition leads to enhanced cell death and reduced survival. Our study demonstrates that one of the earliest events evoked by T-type channel inhibition is a deregulation of pro-survival pathway PI3K/Akt/mTOR and activation of pro-apoptotic stress-activated p38MAPK pathway. The results support the idea of T-type Ca2+ channel as a novel molecular target for antitumor therapy and provide a rational and plausible biological mechanism responsible for the effects induced by T-type Ca2+ channel antagonists in cancer cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A239. Citation Format: Barbara Dziegielewska, Nicholas C.K. Valerie, Amol S. Hosing, James M. Larner, David L. Brautigan, Lloyd S. Gray, Jaroslaw Dziegielewski. T-type calcium channels as a novel molecular target for tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A239.

The anti-inflammatory drug celecoxib inhibits t-type Ca2+ currents in spermatogenic cells yet it elicits the acrosome reaction in mature sperm

Celecoxib (Cx), an anti-inflammatory drug designed to inhibit COX2, can affect some ion channels. T-type (CaV3) channels have been implicated in sperm physiology. Here we report and characterize the Cx induced inhibition of T-type channels in mouse spermatogenic cells. Unexpectedly, Cx can also induce the acrosome reaction (AR), an intracellular Ca2+ ([Ca2+]i) increase and a sperm depolarization. This [Ca2+]i increase possibly results from the ability Cx has to alkalinize intracellular pH (pHi), which is known to activate the sperm specific Ca2+ channel CatSper. As the Cx induced [Ca2+]i increase is sensitive to mibefradil, a CatSper blocker, this channel may mediate the Cx-induced Ca2+ entry leading to the AR. Our observations demonstrate that Cx can compromise fertilization.

Functional and pharmacological consequences of the distribution of voltage‐gated calcium channels inthe renal blood vessels

Calcium channel blockers are widely used to treat hypertension because they inhibit voltage-gated calcium channels that mediate transmembrane calcium influx in, for example, vascular smooth muscle and cardiomyocytes. The calcium channel family consists of several subfamilies, of which the L-type is usually associated with vascular contractility. However, the L-, T- and P-/Q-types of calcium channels are present in the renal vasculature and are differentially involved in controlling vascular contractility, thereby contributing to regulation of kidney function and blood pressure. In the preglomerular vascular bed, all the three channel families are present. However, the T-type channel is the only channel in cortical efferent arterioles which is in contrast to the juxtamedullary efferent arteriole, and that leads to diverse functional effects of L- and T-type channel inhibition. Furthermore, by different mechanisms, T-type channels may contribute to both constriction and dilation of the arterioles. Finally, P-/Q-type channels are involved in the regulation of human intrarenal arterial contractility. The calcium blockers used in the clinic affect not only L-type but also P-/Q- and T-type channels. Therefore, the distinct effect obtained by inhibiting a given subtype or set of channels under experimental settings should be considered when choosing a calcium blocker for treatment. T-type channels seem to be crucial for regulating the GFR and the filtration fraction. Use of blockers is expected to lead to preferential efferent vasodilation, reduction of glomerular pressure and proteinuria. Therefore, renovascular T-type channels might provide novel therapeutic targets, and may have superior renoprotective effects compared to conventional calcium blockers.

Myogenic tone is impaired at low arterial pressure in mice deficient in the low‐voltage‐activated CaV3.1 T‐type Ca2+ channel

Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80mmHg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels.

P2‐083: Age‐related reductions in T‐type calcium channels and their contribution to Alzheimer's disease pathogenesis

Over the past 20 years aberrant calcium dysregulation has been consistently implicated in Alzheimer's disease (AD), either in the initiation of AD, the progression of AD-related pathologies, or in mediating the neuronal and synaptic loss resulting from AD pathology. While many studies have implicated high voltage-gated calcium channels in the calcium dyshomeostasis frequently observed in AD, low voltage-gated T-type calcium channels remained to be investigated. We therefore assessed steady state protein levels of CaV 3.1 in 3, 18, and 24 month-old wild-type mice and found that the channels were robustly reduced with age. To understand the effects of reduced T-type channel levels in the aged brain on the pathogenesis of AD, we treated 3xTg-AD mice with a specific T-type channel blocker, NNC-55-0396, to mimic the age-related reductions. Two groups of female 3xTg-AD mice between the ages of 14 and 16 months were injected intraperitoneally every other day for two weeks with either NNC-55-0396 (20 mg/kg) or vehicle and then sacrificed. Despite an experimental period of just two weeks, in mice with established pathology, we found significant increases in soluble levels of Aβ1-42 with T-type channel inhibition. In addition, insoluble levels of Aβ 1-42 trended toward an increase. Accordingly, Western blotting revealed decreases in C83 and the constitutive α -secretase ADAM10, suggesting that blockade of T-type channels downregulates the non-amyloidogenic processing pathway of APP. Notably, no significant changes in tau pathology were observed. However, the cleavage of p35 to p25, which is carried out by calpains, was dramatically reduced with treatment. Further investigation revealed that calpain 1 and 2 protein levels were decreased with drug treatment. Another substrate of calpains, spectrin, was also analyzed in these samples and was found to be similarly decreased in drug-treated mice compared to controls, suggesting that T-type calcium currents contribute substantially to the regulation of calpains. From our results, we conclude that age-related reductions in T-type calcium channels may have the potential to push APP processing toward the amyloidogenic pathway. This could, in turn, predispose the aging brain to develop Alzheimer's disease.

Calcium-dependent inhibition of T-type calcium channels by TRPV1 activation in rat sensory neurons

We studied the inhibitory effects of transient receptor potential vanilloid-1 (TRPV1) activation by capsaicin on low-voltage-activated (LVA, T-type) Ca(2+) channel and high-voltage-activated (HVA; L, N, P/Q, R) currents in rat DRG sensory neurons, as a potential mechanism underlying capsaicin-induced analgesia. T-type and HVA currents were elicited in whole-cell clamped DRG neurons using ramp commands applied before and after 30-s exposures to 1μM capsaicin. T-type currents were estimated at the first peak of the I-V characteristics and HVA at the second peak, occurring at more positive potentials. Small and medium-sized DRG neurons responded to capsaicin producing transient inward currents of variable amplitudes, mainly carried by Ca(2+). In those cells responding to capsaicin with a large Ca(2+) influx (59% of the total), a marked inhibition of both T-type and HVA Ca(2+) currents was observed. The percentage of T-type and HVA channel inhibition was prevented by replacing Ca(2+) with Ba(2+) during capsaicin application or applying high doses of intracellular BAPTA (20mM), suggesting that TRPV1-mediated inhibition of T-type and HVA channels is Ca(2+)-dependent and likely confined to membrane nano-microdomains. Our data are consistent with the idea that TRPV1-induced analgesia may derive from indirect inhibition of both T-type and HVA channels which, in turn, would reduce the threshold of nociceptive signals generation (T-type channel inhibition) and nociceptive synaptic transmission (HVA-channels inhibition).

In Vitro Characterization of T-Type Calcium Channel Antagonist TTA-A2 and In Vivo Effects on Arousal in Mice

T-type calcium channels have been implicated in many behaviorally important neurophysiological processes, and altered channel activity has been linked to the pathophysiology of neurological disorders such as insomnia, epilepsy, Parkinson's disease, depression, schizophrenia, and pain. We have previously identified a number of potent and selective T-type channel antagonists (Barrow et al., 2007; Shipe et al., 2008; Yang et al., 2008). Here we describe the properties of the antagonist TTA-A2 [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide], assessed in patch-clamp experiments. TTA-A2 blocks T-type channels (Ca(v)3.1, 3.2, 3.3) voltage dependently and with high potency (IC(50) ∼100 nM). Stimulation at 3 Hz revealed additional use dependence of inhibition. A hyperpolarized shift of the channel availability curve and delayed channel recovery from inactivation suggest that the compound preferentially interacts with and stabilizes inactivated channels. The compound showed a ∼300-fold selectivity for Ca(v)3 channels over high-voltage activated calcium channels. Inhibitory effects on native T-type currents were confirmed in brain slice recordings from the dorsal lateral geniculate nucleus and the subthalamic nucleus. Furthermore, we demonstrate that in vivo T-type channel inhibition by TTA-A2 suppresses active wake and promotes slow-wave sleep in wild-type mice but not in mice lacking both Ca(v)3.1 and Ca(v)3.3, suggesting the selective effect of TTA-A2 on recurrent thalamocortical network activity. The discovery of the potent and selective T-type channel antagonist TTA-A2 has enabled us to study the in vivo effects of pharmacological T-channel inhibition on arousal in mice, and it will help to explore the validity of these channels as potential drug targets for sleep-related and other neurological diseases.