T-type Channels Research Articles

Voltage-gated calcium channels generate blastema Ca 2+ fluxes restraining zebrafish fin regenerative outgrowth.

Adult zebrafish fins regenerate to their original size regardless of damage extent, providing a tractable model of organ size and scale control. Gain-of-function of voltage-gated K + channels expressed in fibroblast-lineage blastema cells promotes excessive fin outgrowth, leading to a long-finned phenotype. Similarly, inhibition of the Ca 2+ -dependent phosphatase calcineurin during regeneration causes dramatic fin overgrowth. However, Ca 2+ fluxes and their potential origins from dynamic membrane voltages have not been explored or linked to fin size restoration. We used fibroblast-lineage GCaMP imaging of regenerating adult fins to identify dynamic and heterogeneous Ca 2+ transients in distal blastema cells. Membrane depolarization of isolated regenerating fin fibroblasts triggered Ca 2+ spikes dependent on voltage-gated Ca 2+ channel activity. Single cell transcriptomics identified the voltage-gated Ca 2+ channels cacna1c (L-type channel), cacna1ba (N-type), and cacna1g (T-type) as candidate mediators of fibroblast-lineage Ca 2+ signaling. Small molecule inhibition revealed L- and/or N-type voltage-gated Ca 2+ channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, cacna1g homozygous mutant zebrafish regenerated extraordinarily long fins due to prolonged outgrowth. The regenerated fins far exceeded their original length but with otherwise normal ray skeletons. Therefore, cacna1g mutants uniquely provide a genetic loss-of-function long-finned model that decouples developmental and regenerative fin outgrowth. Live GCaMP imaging of regenerating fins showed T-type Cacna1g channels enable Ca 2+ dynamics in distal fibroblast-lineage blastemal mesenchyme during the outgrowth phase. We conclude "bioelectricity" for fin size control likely entirely reflects voltage-modulated Ca 2+ dynamics in fibroblast-lineage blastemal cells that specifically and steadily decelerates outgrowth at a rate tuned to restore the original fin size.

Interleukin-22 receptor 1-mediated stimulation of T-type Ca2+ channels enhances sensory neuronal excitability through the tyrosine-protein kinase Lyn-dependent PKA pathway

BackgroundInterleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24’s role in peripheral nociception remain unclear.MethodsUsing patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels).ResultsIL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2.ConclusionOur findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.

G protein β subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus

Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gβ2 as an interaction partner. Subsequent assays revealed that the interaction of Gβ2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789–1810) as the Gβ2 interaction site. Coexpression studies of rat Cav3.3 with various Gβγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gβ2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gβ2γ2, but were mimicked by Gβ2 alone as well as other Gβγ dimers, with similar potencies. Deletion of the Gβ2 interaction site abolished the effects of Gβ2γ2. Importantly, these Gβ2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gβ(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gβ interaction with the Cav3.3 C-terminus.

Role of L-type Ca2+ channels in the differential response to oxytocin of uteri from virgin and proven breeder rats

Introduction: The experience of pregnancy affects uterine function well beyond delivery. For instance, the likelihood of accelerated parturitions, spontaneous delivery, and risk of hemorrhage in future pregnancies is increased with parity. While epidemiological evidence for these phenomena is abundant, mechanistic explanations of these and other changes are still being investigated. We previously demonstrated that the response to oxytocin is more robust in the uteri of proven breeder rats and that T-type calcium channels, although slightly more expressed in proven breeder samples had a rather limited impact in this mechanism. Hypothesis: Here, we tested the hypothesis that voltage gated L-type calcium channels contribute more significantly than T-type calcium channels in the differential response to oxytocin in the myometrium from virgin (V) and proven breeder (PB) rats. Methods: V and PB non pregnant female 18-week-old rats were euthanized at proestrus. Uterine strips were suspended in a tissue bath containing Krebs solution (in mM, NaCl 117, KCl 4.7, NaHCO3 25, MgCl2 1.2, KH2PO4 1.2, CaCl2 2.5, glucose 11, pH= 7.4) at 37°C in 95% O2 and 5% CO2. Dose response curves to the L-type calcium channel blocker Verapamil (10E-8 to 10E-5 M) and dose responses to oxytocin in the presence of either 10 E-6 or 10E-5 Verapamil were recorded. Area under the curve, frequency, amplitude, and duration of contractions were measured. L-type channels expression was evaluated with qRT-PCR. T-tests and ANOVA two ways were used for statistical analysis. Results: Compared to control, both doses of Verapamil suppressed 100% of spontaneous and 50% of oxytocin-evoked contractility in V and PB samples. The maximal response to oxytocin was significantly stronger in PB than in V (p<0.001). Thus, blocking L-type Ca2+ channels does not alter the differential response to oxytocin observed in the absence of verapamil. The difference in Expression of the L-type calcium channel revealed a 2-fold increase in PB compared to V uteri. These findings closely align with oxytocin dose response data in the presence of the T-type calcium channels blocker Mibefradil and with the T-type channels expression. Conclusions: Neither L-type or T-type Ca2+ channels appear to be key players in the differential response to oxytocin of V and PB uteri. Instead, it is more likely that other elements of the intracellular calcium control are involved in this phenomenon. Acknowledgements: This study was supported by a Kenneth Suarez Fellowship to RP and an MWU intramural fund to MP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

T-Type Ca2+ Channels Mediate a Critical Period of Plasticity in Adult-Born Granule Cells.

Adult-born granule cells (abGCs) exhibit a transient period of elevated synaptic plasticity that plays an important role in hippocampal function. Various mechanisms have been implicated in this critical period for enhanced plasticity, including minimal GABAergic inhibition and high intrinsic excitability conferred by T-type Ca2+ channels. Here we assess the contribution of synaptic inhibition and intrinsic excitability to long-term potentiation (LTP) in abGCs of adult male and female mice using perforated patch recordings. We show that the timing of critical period plasticity is unaffected by intact GABAergic inhibition such that 4-6-week-old abGCs exhibit LTP that is absent by 8 weeks. Blocking GABAA receptors, or partial blockade of GABA release from PV and nNos-expressing interneurons by a µ-opioid receptor agonist, strongly enhances LTP in 4-week-old GCs, suggesting that minimal inhibition does not underlie critical period plasticity. Instead, the closure of the critical period coincides with a reduction in the contribution of T-type Ca2+ channels to intrinsic excitability, and a selective T-type Ca2+ channel antagonist prevents LTP in 4-week-old but not mature GCs. Interestingly, whole-cell recordings that facilitate T-type Ca2+ channel activity in mature GCs unmasks LTP (with inhibition intact) that is also sensitive to a T-type Ca2+ channel antagonist, suggesting T-type channel activity in mature GCs is suppressed by native intracellular signaling. Together these results show that abGCs use T-type Ca2+ channels to overcome inhibition, providing new insight into how high intrinsic excitability provides young abGCs a competitive advantage for experience-dependent synaptic plasticity.

Functional, Structural and Proteomic Effects of Ageing in Resistance Arteries.

The normal ageing process affects resistance arteries, leading to various functional and structural changes. Systolic hypertension is a common occurrence in human ageing, and it is associated with large artery stiffening, heightened pulsatility, small artery remodeling, and damage to critical microvascular structures. Starting from young adulthood, a progressive elevation in the mean arterial pressure is evidenced by clinical and epidemiological data as well as findings from animal models. The myogenic response, a protective mechanism for the microcirculation, may face disruptions during ageing. The dysregulation of calcium entry channels (L-type, T-type, and TRP channels), dysfunction in intracellular calcium storage and extrusion mechanisms, altered expression of potassium channels, and a change in smooth muscle calcium sensitization may contribute to the age-related dysregulation of myogenic tone. Flow-mediated vasodilation, a hallmark of endothelial function, is compromised in ageing. This endothelial dysfunction is related to increased oxidative stress, lower nitric oxide bioavailability, and a low-grade inflammatory response, further exacerbating vascular dysfunction. Resistance artery remodeling in ageing emerges as a hypertrophic response of the vessel wall that is typically observed in conjunction with outward remodeling (in normotension), or as inward hypertrophic remodeling (in hypertension). The remodeling process involves oxidative stress, inflammation, reorganization of actin cytoskeletal components, and extracellular matrix fiber proteins. Reactive oxygen species (ROS) signaling and chronic low-grade inflammation play substantial roles in age-related vascular dysfunction. Due to its role in the regulation of vascular tone and structural proteins, the RhoA/Rho-kinase pathway is an important target in age-related vascular dysfunction and diseases. Understanding the intricate interplay of these factors is crucial for developing targeted interventions to mitigate the consequences of ageing on resistance arteries and enhance the overall vascular health.

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.

T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and − 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs.9rDzNFNpqNfiW7CgYpAk_tVideo

Allosteric mechanism of CaV3.1 inhibition by Z944, a selective T-type channel antagonist

Allosteric mechanism of CaV3.1 inhibition by Z944, a selective T-type channel antagonist

The T-type calcium channelosome.

T-type calcium channels perform crucial physiological roles across a wide spectrum of tissues, spanning both neuronal and non-neuronal system. For instance, they serve as pivotal regulators of neuronal excitability, contribute to cardiac pacemaking, and mediate the secretion of hormones. These functions significantly hinge upon the intricate interplay of T-type channels with interacting proteins that modulate their expression and function at the plasma membrane. In this review, we offer a panoramic exploration of the current knowledge surrounding these T-type channel interactors, and spotlight certain aspects of their potential for drug-based therapeutic intervention.

EXTH-41. COMBINATION TREATMENT OF CALCIUM CHANNEL ANTAGONIST MIBEFRADIL ENHANCES THE EFFECT OF HYDROXYUREA AT THERAPEUTIC RANGES IN HIGH-GRADE MENINGIOMA IN VITRO

Abstract High-grade meningiomas (MNs), WHO Grade II- III, are highly aggressive brain tumors that are difficult to treat. Standard chemotherapy agents are, at best, moderately effective in treating high-grade MNs. There is great interest in developing combinatorial drug treatment regimens to enhance the effects of standard chemotherapy. Many cancer types, including brain tumors, have an increase in the expression of voltage-gated calcium channels (VGCCs). These channels are involved in the regulation of cell cycle, proliferation, and apoptosis, among other functions. FDA-approved VGCC antagonists nimodipine (NIMO) and mibefradil (MIB) target two different VGCCs, L- and T-type channels, and have been of great interest for their use in combination therapy in other cancers. We sought to determine the efficacy of chemotherapy agents tested at clinically relevant concentrations for the treatment of high-grade MNs alone or in combination with NIMO or MIB. We cultured CH-157MN and IOMM-Lee MN cells and assessed their cell survival, apoptosis, and proliferation in the presence of temozolomide, octreotide, sunitinib, or hydroxyurea (HU). We confirmed that HU (150 µM) was the most efficacious chemotherapy agent, decreasing cell viability at therapeutic ranges. Monotherapy treatment with 2 or 4 µM MIB significantly decreased cell viability in both cell lines, whereas 1 µM NIMO only affected IOMM-Lee cells. The combination of HU and MIB further decreased cell viability but only in the most aggressive cell line, IOMM-Lee, whereas NIMO did not enhance HU’s ability to decrease cell survival. Remarkably, we detect an enhancement of HU’s effect by MIB when both agents are used within therapeutic ranges, as reported by pharmacokinetic studies. Here we demonstrate for the first time that T-type calcium channels play an important role in MN survival. The combination of HU and MIB suggests their use could be a promising treatment for high-grade MN.

Calcium channel blockers for hypertension: old, but still useful

Calcium channel blockers (CCBs) constitute a heterogeneous class of drugs that can be divided into dihydropyridines (DHPs) and non-DHPs. DHP-CCBs are subcategorized into four generations based on the duration of activity and pharmacokinetics, while non-DHP-CCBs are subcategorized into phenylethylamine and benzodiazepine derivatives. DHP-CCBs are vascular-selective and function as potent vasodilators, whereas non-DHP-CCBs are cardiac-selective and are useful for treating tachyarrhythmia, but reduce cardiac contractility and heart rate. Traditional DHP-CCBs (nifedipine) mainly block L-type calcium channels, whereas novel CCBs block N-type (amlodipine) and/or T-type channels (efonidipine) in addition to L-type channels, leading to organ-protective effects. DHP-CCBs have a potent blood pressure–lowering effect and suppress atherosclerosis and coronary vasospasm. Diltiazem, a non-DHP-CCB, is highly effective for vasospasm control. CCBs reduce left ventricular hypertrophy and arterial stiffness. Amlodipine, a DHP-CCB, reduces blood pressure variability. L/N- and L/T-type CCBs combined with renin-angiotensin system blockers reduce proteinuria and improve kidney function compared with L-type CCBs. According to large-scale trials, DHP-CCBs reduce cardiovascular events in patients with isolated systolic hypertension, as well as in elderly and high-risk patients. Accordingly, CCBs are indicated for hypertension in elderly patients, isolated systolic hypertension, angina pectoris, and coronary vasospasm. Non-DHP-CCBs are contraindicated in high-grade heart block, bradycardia (<60 beats per minute [bpm]), and heart failure with reduced ejection fraction (HFrEF). DHP-CCBs should be used with caution in patients with tachyarrhythmia, HFrEF, and severe leg edema, and non-DHP-CCBs should be used carefully in those with constipation. Each CCB has distinct pharmacokinetics and side effects, underscoring the need for meticulous consideration in clinical practice.

Harmaline toxicity on dorsal striatal neurons and its role in tremor

Harmaline is one of the β-carboline derivative compounds that is widely distributed in the food chain and human tissues. Harmine, a dehydrogenated form of harmaline, appeared to have a higher concentration in the brain, and appeared to be elevated in essential tremor (ET) and Parkinson’s disease. Exogenous harmaline exposure in high concentration has myriad consequences, including inducing tremor, and causing neurodegeneration of Purkinje cells in the cerebellum. Harmaline-induced tremor is an established animal model for human ET, but its underlying mechanism is still controversial. One hypothesis posits that the inferior olive-cerebellum pathway is involved, and CaV3.1 T-type Ca2+ channel is a critical target of action. However, accumulating evidence indicates that tremor can be generated without disturbing T-type channels. This implies that additional neural circuits or molecular targets are involved. Using in vitro slice Ca2+-imaging and patch clamping, we demonstrated that harmaline reduced intracellular Ca2+ and suppressed depolarization-induced spiking activity of medium spiny striatal neurons (MSN), and this effect of harmaline can be partially attenuated by sulpiride (5 µM). In addition, the frequencies of spontaneous excitatory post-synaptic currents (sEPSCs) on MSNs were also significantly attenuated. Furthermore, the induced tremor in C57BL/6 J mice by harmaline injections (i.p. 12.5–18 mg/kg) was also shown to be attenuated by sulpiride (20 mg/kg). This series of experiments suggests that the dorsal striatum is a site of harmaline toxic action and might contribute to tremor generation. The findings also provide evidence that D2 signaling might be a part of the mechanism underlying essential tremor.

Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice

BackgroundAdipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons.ResultsAdiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCβ1). Blocking PKCβ1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCβ1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2.ConclusionOur study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCβ1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

T-type calcium channel modulation by hydrogen sulfide in neuropathic pain conditions.

Neuropathic pain can appear as a direct or indirect nerve damage lesion or disease that affects the somatosensory nervous system. If the neurons are damaged or indirectly stimulated, immune cells contribute significantly to inflammatory and neuropathic pain. After nerve injury, peripheral macrophages/spinal microglia accumulate around damaged neurons, producing endogenous hydrogen sulfide (H2S) through the cystathionine-γ-lyase (CSE) enzyme. H2S has a pronociceptive modulation on the Cav3.2 subtype, the predominant Cav3 isoform involved in pain processes. The present review provides relevant information about H2S modulation on the Cav3.2 T-type channels in neuropathic pain conditions. We have discussed that the dual effect of H2S on T-type channels is concentration-dependent, that is, an inhibitory effect is seen at low concentrations of 10µM and an augmentation effect on T-current at 100µM. The modulation mechanism of the Cav3.2 channel by H2S involves the direct participation of the redox/Zn2+ affinity site located in the His191 in the extracellular loop of domain I of the channel, involving a group of extracellular cysteines, comprising C114, C123, C128, and C1333, that can modify the local redox environment. The indirect interaction pathways involve the regulation of the Cav3.2 channel through cytokines, kinases, and post-translational regulators of channel expression. The findings conclude that the CSE/H2S/Cav3.2 pathway could be a promising therapeutic target for neuropathic pain disorders.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 μM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 μM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

Inhibition of T-Type Calcium Channels With TTA-P2 Reduces Chronic Neuropathic Pain Following Spinal Cord Injury in Rats

Spinal cord injury (SCI)-induced neuropathic pain (SCI-NP) develops in up to 60 to 70% of people affected by traumatic SCI, leading to a major decline in quality of life and increased risk for depression, anxiety, and addiction. Gabapentin and pregabalin, together with antidepressant drugs, are commonly prescribed to treat SCI-NP, but their efficacy is unsatisfactory. The limited efficacy of current pharmacological treatments for SCI-NP likely reflects our limited knowledge of the underlying mechanism(s) responsible for driving the maintenance of SCI-NP. The leading hypothesis in the field supports a major role for spontaneously active injured nociceptors in driving the maintenance of SCI-NP. Recent data from our laboratory provided additional support for this hypothesis and identified the T-type calcium channels as key players in driving the spontaneous activity of SCI-nociceptors, thus providing a rational pharmacological target to treat SCI-NP. To test whether T-type calcium channels contribute to the maintenance of SCI-NP, male and female SCI and sham rats were treated with TTA-P2 (a blocker of T-type calcium channels) to determine its effects on mechanical hypersensitivity (as measured with the von Frey filaments) and spontaneous ongoing pain (as measured with the conditioned place preference paradigm), and compared them to the effects of gabapentin, a blocker of high voltage–activated calcium channels. We found that both TTA-P2 and gabapentin reduced mechanical hypersensitivity in male and females SCI rats, but surprisingly only TTA-P2 reduced spontaneous ongoing pain in male SCI rats. PerspectivesSCI-induced neuropathic pain, and in particular the spontaneous ongoing pain component, is notoriously very difficult to treat. Our data provide evidence that inhibition of T-type calcium channels reduces spontaneous ongoing pain in SCI rats, supporting a clinically relevant role for T-type channels in the maintenance of SCI-induced neuropathic pain.

The role of T-type channels in the differential response to oxytocin of virgin and proven breeder rats uteri

Introduction: It is commonly known that an increase in parity is associated with faster labor times, increased risk of hemorrhage, and greater chance of spontaneous delivery (1). The mechanisms of these differences have not yet been fully elucidated. Earlier research conducted in the lab demonstrated that pregnancy alters the response of uterine muscles to oxytocin. Hypothesis: We hypothesized that the intracellular calcium handling machinery, particularly T-type calcium channels, contributes to the differential response to oxytocin in the myometrium from virgin (V) and proven breeder (PB) rats. Methods: Virgin (V) and proven breeder (PB) NP female 18-week-old rats were staged and euthanized at proestrus. One uterine horn was dissected in strips suspended in a tissue bath containing Krebs solution (in mM, NaCl 117, KCl 4.7, NaHCO3 25, MgCl2 1.2, KH2PO4 1.2, CaCl2 2.5, glucose 11, pH= 7.4) at 37°C and bubbled with 95% O2 and 5% CO2. Dose response curves to oxytocin (10E-10 to 10E-5 M) were recorded in the presence of the T-type calcium channels inhibitor Mibefradil. Contractility was evaluated as the area under the curve of recorded contractions. The other uterine horn was flash frozen in liquid NO2 and later used for qRT-PCR experiments. T-tests and ANOVA two ways were used for statistical analysis. Results: Mibefradil significantly reduced the spontaneous contractility of the sample and the response to oxytocin (P<0.001). The maximal contractility in the two groups was similar, whereas in the experiments without the drug PB max contractility was higher. However, the range of activation (Max-Min) was higher for PB samples, as previously observed in the absence of the drug. RT-PCR revealed a two-fold increase in T-type channel receptors on the PB uterus compared to the V. Conclusions: Although T-type channels are slightly more expressed in PB than in V uterine tissue, their impact on the differential response to Oxytocin mechanism is limited. Thus, L-type Calcium channels seem to play a more important role in this mechanism. This study was supported by a Kenneth Suarez Fellowship to EB and an MWU intramural fund to MP This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Volume of Fluid Simulations of Heat Transfer in Liquid-Liquid Segmented Flows in Microfluidic channel

This study presents the investigations on the effect of heat transfer on droplet formation in T-type microfluidic channel. Mineral oil acts as a continuous phase, and water acts as a dispersed phase. The Volume of Fluid model is used to investigate the formation of droplets of water in oil in the microchannel. The physical properties of both fluids change with an increase in temperature of dispersed phase, of which the dominant properties are viscosities of fluids and interfacial tension. The parameters such as droplet diameter, distance between two consecutive droplets and detachment time were studied with respect to temperature in the range of 300 K to 325 K. These properties are also studied at different flow rate ratios 0.125, 0.25, and 0.5. The distance between two consecutive droplets was found to decrease with the increase in temperature due to the rapid motion of droplets in the temperature zone. The droplet detachment time also decreases, due to the decrease in interfacial forces which hold the droplets in the dispersed phase. The droplet diameter decreases initially with the increase in temperature, but it was observed that after 320 K the diameter of the droplet increases due to the coalescence of two consecutive droplets downstream in the microchannel. This temperature is reported as a limiting condition for thermally controlled water-in-oil microemulsions.