Antihypertensive Effects of Lindera erythrocarpa Makino via NO/cGMP Pathway and Ca2+ and K+ Channels.

Studies have demonstrated the therapeutic effects of Lindera plants. This study was undertaken to reveal the antihypertensive properties of Lindera erythrocarpa leaf ethanolic extract (LEL). Aorta segments of Sprague-Dawley rats were used to study the vasodilatory effect of LEL, and the mechanisms involved were evaluated by treating specific inhibitors or activators that affect the contractility of blood vessels. Our results revealed that LEL promotes a vasorelaxant effect through the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway, blocking the Ca2+ channels, opening the K+ channels, and inhibiting the vasoconstrictive action of angiotensin II. In addition, the effects of LEL on blood pressure were investigated in spontaneously hypertensive rats by the tail-cuff method. LEL (300 or 1000 mg/kg) was orally administered to the rats, and 1000 mg/kg of LEL significantly lowered the blood pressure. Systolic blood pressure decreased by -20.06 ± 4.87%, and diastolic blood pressure also lowered by -30.58 ± 5.92% at 4 h in the 1000 mg/kg LEL group. Overall, our results suggest that LEL may be useful to treat hypertensive diseases, considering its vasorelaxing and hypotensive effects.

ER-plasma membrane contact sites deliver ER lipids and proteins for rapid cell surface expansion.

As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by ∼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2.

Tyrosine phosphorylation and palmitoylation of TRPV2 ion channel tune microglial beta-amyloid peptide phagocytosis.

Alzheimer's disease (AD) is the leading form of dementia, characterized by the accumulation and aggregation of amyloid in brain. Transient receptor potential vanilloid 2 (TRPV2) is an ion channel involved in diverse physiopathological processes, including microglial phagocytosis. Previous studies suggested that cannabidiol (CBD), an activator of TRPV2, improves microglial amyloid-ß (Aß) phagocytosis by TRPV2 modulation. However, the molecular mechanism of TRPV2 in microglial Aß phagocytosis remains unknown. In this study, we aimed to investigate the involvement of TRPV2 channel in microglial Aß phagocytosis and the underlying mechanisms. Utilizing human datasets, mouse primary neuron and microglia cultures, and AD model mice, to evaluate TRPV2 expression and microglial Aß phagocytosis in both in vivo and in vitro. TRPV2 was expressed in cortex, hippocampus, and microglia.Cannabidiol (CBD) could activate and sensitize TRPV2 channel. Short-term CBD (1 week) injection intraperitoneally (i.p.) reduced the expression of neuroinflammation and microglial phagocytic receptors, but long-term CBD (3 week) administration (i.p.) induced neuroinflammation and suppressed the expression of microglial phagocytic receptors in APP/PS1 mice. Furthermore, the hyper-sensitivity of TRPV2 channel was mediated by tyrosine phosphorylation at the molecular sites Tyr(338), Tyr(466), and Tyr(520) by protein tyrosine kinase JAK1, and these sites mutation reduced the microglial Aß phagocytosis partially dependence on its localization. While TRPV2 was palmitoylated at Cys 277 site and blocking TRPV2 palmitoylation improved microglial Aß phagocytosis. Moreover, it was demonstrated that TRPV2 palmitoylation was dynamically regulated by ZDHHC21. Overall, our findings elucidated the intricate interplay between TRPV2 channel regulated by tyrosine phosphorylation/dephosphorylation and cysteine palmitoylation/depalmitoylation, which had divergent effects on microglial Aß phagocytosis. These findings provide valuable insights into the underlying mechanisms linking microglial phagocytosis and TRPV2 sensitivity, and offer potential therapeutic strategies for managing AD.

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind CaV2 calcium channel C-termini in vitro.

PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 chanels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly bound the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint.

Ca2+ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity.

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (Pr). Surprisingly, VGCC levels are highly predictive of heterogeneous Pr among individual synapses of either low- or high-Pr inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-Pr synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ-3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-Pr inputs, yet positively correlate with Pr among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses.

Determinants of interactions of a novel next-generation gabapentinoid NVA1309 and mirogabalin with the Cavα2δ-1 subunit.

NVA1309 is a non-brain penetrant next-generation gabapentinoid shown to bind Cavα2δ at R243 within a triple Arginine motif forming the binding site for gabapentin and pregabalin. In this study we have compared the effects of NVA1309 with Mirogabalin, a gabapentinoid drug with higher affinity for the voltage-gated calcium channel subunit Cavα2δ-1 than pregabalin which is approved for post-herpetic neuralgia in Japan, Korea and Taiwan. Both NVA1309 and mirogabalin inhibit Cav2.2 currents in vitro and decrease Cav2.2 plasma membrane expression with higher efficacy than pregabalin. Mutagenesis of the classical binding residue arginine R243 and the newly identified binding residue lysine K615 reverse the effect of mirogabalin on Cav2.2 current, but not that of NVA1309.

Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation.

Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.

Mitochondrial calcium uniporter channel gatekeeping in cardiovascular disease.

The mitochondrial calcium (mCa2+) uniporter channel (mtCU) resides at the inner mitochondrial membrane and is required for Ca2+ to enter the mitochondrial matrix. The mtCU is essential for cellular function, as mCa2+ regulates metabolism, bioenergetics, signaling pathways and cell death. mCa2+ uptake is primarily regulated by the MICU family (MICU1, MICU2, MICU3), EF-hand-containing Ca2+-sensing proteins, which respond to cytosolic Ca2+ concentrations to modulate mtCU activity. Considering that mitochondrial function and Ca2+ signaling are ubiquitously disrupted in cardiovascular disease, mtCU function has been a hot area of investigation for the last decade. Here we provide an in-depth review of MICU-mediated regulation of mtCU structure and function, as well as potential mtCU-independent functions of these proteins. We detail their role in cardiac physiology and cardiovascular disease by highlighting the phenotypes of different mutant animal models, with an emphasis on therapeutic potential and targets of interest in this pathway.

Integrated single-cell RNA-seq analysis reveals mitochondrial calcium signaling as a modulator of endothelial-to-mesenchymal transition.

Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target.

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells.

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Orai1 and Orai3 act through distinct signalling axes to promote stemness and tumorigenicity of breast cancer stem cells.

BACKGROUND: One of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood. METHODS: A highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo. RESULTS: Orai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis. CONCLUSION: We found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.

Natural allelic diversity of the calcium signaling regulators in plants.

Calcium ions act as secondary messengers in diverse signaling pathways in plants throughout their life cycle. Studies have revealed that calcium is involved in developmental events and in responses to external stimuli, such as biotic and abiotic stresses. Cellular calcium ion levels are tightly controlled by intricate molecular machinery such as calcium channels and pumps. Transient and spatial fluctuations in calcium levels are subsequently recognized by diverse calcium-decoding molecules, resulting in signal transduction. In this review, we highlight recent findings on natural variations in genes controlling calcium signaling in diverse plant biological processes. We then show how the calcium ion context is utilized by fine-tuning the natural variation in centrally important genes.

Distinct active zone protein machineries mediate Ca2+ channel clustering and vesicle priming at hippocampal synapses.

Action potentials trigger neurotransmitter release at the presynaptic active zone with spatiotemporal precision. This is supported by protein machinery that mediates synaptic vesicle priming and clustering of CaV2 Ca2+ channels nearby. One model posits that scaffolding proteins directly tether vesicles to CaV2s; however, here we find that at mouse hippocampal synapses, CaV2 clustering and vesicle priming are executed by separate machineries. CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins but distinct interaction motifs independently execute these functions. In transfected cells, Liprin-α and RIM form co-assemblies that are separate from CaV2-organizing complexes. At synapses, Liprin-α1-Liprin-α4 knockout impairs vesicle priming but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering CaV2s. We conclude that active zones consist of distinct machineries to organize CaV2s and prime vesicles, and Liprin-α and PTPσ specifically support priming site assembly.

The role of calcium channels in osteoporosis and their therapeutic potential.

Osteoporosis, a systemic skeletal disorder marked by diminished bone mass and compromised bone microarchitecture, is becoming increasingly prevalent due to an aging population. The underlying pathophysiology of osteoporosis is attributed to an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Osteoclasts play a crucial role in the development of osteoporosis through various molecular pathways, including the RANK/RANKL/OPG signaling axis, cytokines, and integrins. Notably, the calcium signaling pathway is pivotal in regulating osteoclast activation and function, influencing bone resorption activity. Disruption in calcium signaling can lead to increased osteoclast-mediated bone resorption, contributing to the progression of osteoporosis. Emerging research indicates that calcium-permeable channels on the cellular membrane play a critical role in bone metabolism by modulating these intracellular calcium pathways. Here, we provide an overview of current literature on the regulation of plasma membrane calcium channels in relation to bone metabolism with particular emphasis on their dysregulation during the progression of osteoporosis. Targeting these calcium channels may represent a potential therapeutic strategy for treating osteoporosis.

The episodic ataxias.

The primary episodic ataxias (EAs) are a group of autosomal-dominant disorders characterized by transient recurrent incoordination and truncal instability, often triggered by physical exertion or emotional stress and variably associated with progressive baseline ataxia. There are now nine designated subtypes EA1-9 (OMIM) and late onset cerebellar ataxia with episodic features as newly designated SCA27B, based largely on genetic loci. Mutations have been identified in multiple individuals and families in 4 of the 9 EA subtypes, mostly with the onset before adulthood. This chapter focuses on the clinical assessment and management of EA, genetic diagnosis, and neurophysiologic consequences of the causative mutations in the best characterized EA syndromes: EA1 caused by mutations in KCNA1 encoding a neuronal voltage-gated potassium channel, EA2 caused by mutations in CACNA1A encoding a neuronal voltage-gated calcium channel, EA6 caused by mutations in SLC1A3 encoding a glutamate transporter that is also an anion channel, and SCA27B with late onset episodic ataxia caused by an intronic trinucleotide repeat in FGF14 encoding fibroblast growth factor 14 important in regulating the distribution of voltage-gated sodium channels in the cerebellar Purkinje and granule cells. The study of EA has illuminated previously unrecognized but important roles of ion channels and transporters in brain function with shared mechanisms underlying cerebellar ataxia, migraine, and epilepsy.

Familial hemiplegic migraine.

Hemiplegic migraine consists of attacks of migraine with aura that includes reversible motor weakness. It is classified as familial or sporadic depending on the involvement or not of a first or second degree relative. The most described subtypes of familial hemiplegic migraine include FHM1, FHM2, and FHM3. These have been demonstrated to have a mutation in either CACNA1A, ATP1A2 or SCN1A, which encode different subunits of channels, involving P/Q-type calcium channel, Na/K pump and Na channel, respectively, located in neurons and glial cells. Mutations localized in different genes are defined as "other loci." Patients with a known mutation can have different genetic penetrance, and may present a more complex and disabling phenotype that develops earlier in life. The clinical manifestations can be similar in the three mutations, including neurologic comorbidities other than muscular weakness, such as episodes of loss of consciousness, epilepsy, gait or limb ataxia or movement disorders, among others. Treatment includes antiepileptics such as lamotrigine, valproate or topiramate, calcium blockers such as flunarizine or verapamil and acetazolamide.

Descending to inhibit: Antagonist-induced downward shift of VSD II in TPC2.

In this issue of Structure, Chi et al.1 report structural and functional studies that reveal the inhibition mechanism of the lysosomal two-pore channel TPC2 by the antagonist SG-094, which is of interest for drug development. Antagonist binding induces the downward displacement of the voltage-sensor domain II (VSD II), which is accompanied by asymmetric conformational rearrangements of the entire channel.

A versatile residue numbering scheme for Nav and Cav channels.

Voltage-gated sodium (Nav) and calcium (Cav) channels are responsible for the initiation of electrical signals. They have long been targeted for the treatment of various diseases. The mounting number of cryoelectron microscopy (cryo-EM) structures for diverse subtypes of Nav and Cav channels from multiple organisms necessitates a generic residue numbering system to establish the structure-function relationship and to aid rational drug design or optimization. Here we suggest a structure-based residue numbering scheme, centering around the most conserved residues on each of the functional segments. We elaborate the generic numbers through illustrative examples, focusing on representative drug-binding sites of eukaryotic Nav and Cav channels. We also extend the numbering scheme to compare common disease mutations among different Nav subtypes. Application of the generic residue numbering scheme affords immediate insights into hotspots for pathogenic mutations and critical loci for drug binding and will facilitate drug discovery targeting Nav and Cav channels.

Mirogabalin: a novel gabapentinoid or another false dawn?

PURPOSE OF REVIEW: Mirogabalin is a novel gabapentinoid medication for the treatment of neuropathic pain. The purpose of this review is to discuss current evidence for its use. Gabapentinoids are widely prescribed for neuropathic pain. Mirogabalin offers theoretical advantages over traditional gabapentinoids due to its specificity for the α2δ-1 subunit of voltage-gated calcium channels. It is theorised that this specificity may reduce adverse drug reactions by minimising binding to the α2δ-2 subunit which is responsible for many of the gabapentinoid side effects. RECENT FINDINGS: Mirogabalin's slower dissociation from the α2δ-1 compared with α2δ-2, and its higher potency may also impart an efficacy benefit over traditional gabapentinoids. These theoretical advantages of mirogabalin remain inconclusive in clinical practice, with mixed evidence regarding mirogabalin versus traditional gabapentinoids. Some studies suggest a reduced side effect profile yet, others fail to demonstrate significant differences. Regarding efficacy, mirogabalin may be superior to placebo for several neuropathic pain syndromes, but evidence of widespread benefit over traditional gabapentinoids is currently lacking. SUMMARY: Mirogabalin offers theoretical promise, but large, independent studies are required to further assess its performance versus traditional gabapentinoids.

Voltage-gated calcium channels act upstream of adenylyl cyclase Ac78C to promote timely initiation of dendrite regeneration.

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.