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Active removal of Na⁺ from the cytosol into the vacuole plays a critical role in salinity tissue tolerance, but another, often neglected component of this trait is Na⁺ retention in vacuoles. This retention is based on an efficient control of Na⁺-permeable slow- and fast-vacuolar channels that mediate the back-leak of Na⁺ into cytosol and, if not regulated tightly, could result in a futile cycle. This Tansley insight summarizes our current knowledge of regulation of tonoplast Na⁺-permeable channels and discusses the energy cost of vacuolar Na⁺ sequestration, under different scenarios. We also report on a phylogenetic and bioinformatic analysis of the plant two-pore channel family and the difference in its structure and regulation between halophytes and glycophytes, in the context of salinity tolerance.

Unknown mechanisms tightly regulate the basal activity of the wound-inducible defence mediator jasmonate (JA) in undamaged tissues. However, the Arabidopsis fatty acid oxygenation upregulated2 (fou2) mutant in vacuolar two-pore channel 1 (TPC1D454N) displays high JA pathway activity in undamaged leaves. This mutant was used to explore mechanisms controlling basal JA pathway regulation. fou2 was re-mutated to generate novel ‘ouf’ suppressor mutants. Patch-clamping was used to examine TPC1 cation channel characteristics in the ouf suppressor mutants and in fou2. Calcium (Ca2+) imaging was used to study the effects fou2 on cytosolic Ca2+ concentrations. Six intragenic ouf suppressors with near wild-type (WT) JA pathway activity were recovered and one mutant, ouf8, affected the channel pore. At low luminal calcium concentrations, ouf8 had little detectable effect on fou2. However, increased vacuolar Ca2+ concentrations caused channel occlusion, selectively blocking K+ fluxes towards the cytoplasm. Cytosolic Ca2+ concentrations in unwounded fou2 were found to be lower than in the unwounded WT, but they increased in a similar manner in both genotypes following wounding. Basal JA pathway activity can be controlled solely by manipulating endomembrane cation flux capacities.We suggest that changes in endomembrane potential affect JA pathway activity.

Calcium ion (Ca(2+) ) signalling triggered by insect herbivory is an intricate network with multiple components, involving positive and negative regulators. Real-time, noninvasive imaging of entire Arabidopsis thaliana rosettes was employed to monitor cytosolic free calcium ([Ca(2+) ]cyt ) elevations in local and systemic leaves in response to wounding and Spodoptera littoralis feeding. Luminescence emitted by the cytosol-localized Ca(2+) reporter aequorin was imaged using a high-resolution photon-counting camera system. Spodoptera littoralis feeding on Arabidopsis induced both local and systemic [Ca(2+) ]cyt elevations. Systemic [Ca(2+) ]cyt signals were found predominantly in adjacent leaves with direct vascular connections to the treated leaf and appeared with a delay of 1 to 2 min. Simulated herbivory by wounding always induced a local [Ca(2+) ]cyt response, but a systemic one only when the midrib was wounded. This systemic [Ca(2+) ]cyt response was suppressed by the presence of insect-derived oral secretions as well as in a mutant of the vacuolar cation channel, Two Pore Channel 1 (TPC1). Our results provide evidence that in Arabidopsis insect herbivory induces both local and systemic [Ca(2+) ]cyt signals that distribute within the vascular system. The systemic [Ca(2+) ]cyt signal could play an important signalling role in systemic plant defence.

Calcium ion (Ca2+) signalling triggered by insect herbivory is an intricate network withmultiple components, involving positive and negative regulators. Real-time, noninvasive imaging of entire Arabidopsis thaliana rosettes was employed to monitor cytosolic free calcium ([Ca2+]cyt) elevations in local and systemic leaves in response to wounding and Spodoptera littoralis feeding. Luminescence emitted by the cytosol-localized Ca2+ reporter aequorin was imaged using a high-resolution photon-counting camera system. Spodoptera littoralis feeding on Arabidopsis induced both local and systemic [Ca2+]cyt elevations. Systemic [Ca2+]cyt signals were found predominantly in adjacent leaves with direct vascular connections to the treated leaf and appeared with a delay of 1 to 2 min. Simulated herbivory by wounding always induced a local [Ca2+]cyt response, but a systemic one only when the midrib was wounded. This systemic [Ca2+]cyt response was suppressed by the presence of insect-derived oral secretions as well as in a mutant of the vacuolar cation channel, Two Pore Channel 1 (TPC1). Our results provide evidence that in Arabidopsis insect herbivory induces both local and systemic [Ca2+]cyt signals that distribute within the vascular system. The systemic [Ca2+]cyt signal could play an important signalling role in systemic plant defence.

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. Sphingosine is a small fat molecule that has been suggested to act as a signal inside cells. Individuals with a rare neurodegenerative disease called Niemann-Pick disease type C accumulate sphingosine and other fat molecules in cell compartments called lysosomes. Intriguingly, this fat accumulation is accompanied by an altered movement of calcium ions in and out of lysosomes. In healthy cells, an increase in calcium ion levels can trigger a process called autophagy, in which proteins and other cell components are destroyed in a controlled manner. This is thought to be caused by the release of calcium ions from lysosomes, which stimulates a protein called TFEB to move into the nucleus of the cell to activate genes involved in autophagy. Two proteins on the surface of lysosomes called TPC1 and TPC2 are believed to act as channels that can release calcium ions from lysosomes. However, it was not clear how sphingosine could disrupt calcium ion movements in patients with Niemann-Pick disease type C. Here, Hoeglinger et al. have used a new approach to understand how calcium ions and sphingosine are linked in both healthy and diseased cells. The experiments use a form of sphingosine called “caged sphingosine” that is only activated when it is exposed to a flash of light, which makes it possible to increase the levels of this molecule in cells in a precise way. Hoeglinger et al. found that sphingosine triggered the release of calcium ions from lysosomes. This release required the TPC1 protein and resulted in TFEB moving into the cell nucleus. Further experiments confirm that sphingosine accumulates in the lysosomes of cells taken from patients with Niemann-Pick disease type C. In these cells, the activation of caged sphingosine resulted in a much smaller release of calcium ions from lysosomes than that observed in healthy cells. Together, Hoeglinger et al.’s findings show that sphingosine acts as a signal to trigger the release of calcium ions from lysosomes, which in turn promotes autophagy. The next challenge is to find out exactly how sphingosine opens the calcium ion channels.

Ca²⁺signatures are central to developmental processes and adaptive responses in plants. However, high‐resolution studies of Ca²⁺dynamics using genetically encoded Ca²⁺indicators (GECIs) such as Yellow Cameleon (YC) proteins have so far not been conducted in important model crops such as rice (Oryza sativa). We conducted a comparative study of 35S and ubiquitin‐10 (UBQ10) promoter functionality in Arabidopsis thaliana and O. sativa plants expressing the Ca²⁺indicator Yellow Cameleon 3.6 (YC3.6) under control of the UBQ10 or 35S promoter. Ca²⁺signatures in roots of both species were analyzed during exposure to hyperpolarization/depolarization cycles or in response to application of the amino acid glutamate. We found a superior performance of the UBQ10 promoter with regard to expression pattern, levels and expression stabilities in both species. We observed remarkable differences between the two species in the spatiotemporal parameters of the observed Ca²⁺signatures. Rice appeared in general to respond with a lower maximal signal amplitude but greatly increased signal duration when compared with Arabidopsis. Our results identify important advantages to using the UBQ10 promoter in Arabidopsis and rice and in T‐DNA mutant backgrounds. Moreover, the observed differences in Ca²⁺signaling in the two species underscore the need for comparative studies to achieve a comprehensive understanding of Ca²⁺signaling in plants.

Calcium ion (Ca 2+ ) signalling triggered by insect herbivory is an intricate network with multiple components, involving positive and negative regulators. Real‐time, noninvasive imaging of entire Arabidopsis thaliana rosettes was employed to monitor cytosolic free calcium ([Ca 2+ ] cyt ) elevations in local and systemic leaves in response to wounding and Spodoptera littoralis feeding. Luminescence emitted by the cytosol‐localized Ca 2+ reporter aequorin was imaged using a high‐resolution photon‐counting camera system. Spodoptera littoralis feeding on Arabidopsis induced both local and systemic [Ca 2+ ] cyt elevations. Systemic [Ca 2+ ] cyt signals were found predominantly in adjacent leaves with direct vascular connections to the treated leaf and appeared with a delay of 1 to 2 min. Simulated herbivory by wounding always induced a local [Ca 2+ ] cyt response, but a systemic one only when the midrib was wounded. This systemic [Ca 2+ ] cyt response was suppressed by the presence of insect‐derived oral secretions as well as in a mutant of the vacuolar cation channel, Two Pore Channel 1 ( TPC 1). Our results provide evidence that in Arabidopsis insect herbivory induces both local and systemic [Ca 2+ ] cyt signals that distribute within the vascular system. The systemic [Ca 2+ ] cyt signal could play an important signalling role in systemic plant defence.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch‐activated K 2P potassium channel TREK‐1 ( KCNK2 or K 2P 2.1). This mutation introduces abnormal sodium permeability to TREK‐1. In addition, mutant channels exhibit a hypersensitivity to stretch‐activation, suggesting that the selectivity filter is directly involved in stretch‐induced activation and desensitization. Increased sodium permeability and stretch‐sensitivity of mutant TREK‐1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT. We present a pharmacological strategy to rescue the selectivity defect of the TREK‐1 pore. Our findings provide important insights for future studies of K 2P channel stretch‐activation and the role of TREK‐1 in mechano‐electrical feedback in the heart. Synopsis A point mutation in the selectivity filter of the stretch‐activated K 2P potassium channel TREK‐1 was identified in a patient with right ventricular outflow tract tachycardia. The mutation most likely causes arrhythmias through abnormal sodium permeability and hypersensitivity to stretch‐activation. Analysis of a patient with right ventricular outflow tract tachycardia (RVOT‐VT) led to the identification of a heterozygous mutation, resulting in an Ile to Thr exchange directly preceding the selectivity filter of the K 2P potassium channel TREK‐1. The mutation introduces an abnormal sodium permeability and a hypersensitivity to stretch‐activation to TREK‐1 channels. The study suggests that the selectivity filter is directly involved in stretch‐induced activation and desensitization of stretch‐sensitive K 2P potassium channels. Increased sodium permeability and stretch‐sensitivity of mutant TREK‐1 channels may trigger arrhythmias in areas of the heart with high physical strain. The findings provide important insights for future studies of K 2P channel stretch‐activation and the role of TREK‐1 in mechano‐electrical feedback in the heart. Graphical Abstract A point mutation in the selectivity filter of the stretch‐activated K 2P potassium channel TREK‐1 was identified in a patient with right ventricular outflow tract tachycardia. The mutation most likely causes arrhythmias through abnormal sodium permeability and hypersensitivity to stretch‐activation.

TASK-1 is a two-pore K + leak channel. The name, TASK-1, stands for TWIK-related acid-sensitive potassium channel 1, and this channel is encoded by the KCNK3 gene. TASK-1 channels are expressed in humans and modulate cell excitability in excitable cells such as neurons, cardiomyocytes, and vascular smooth muscle cells. TASK-1 inhibition is a mechanism of action for some respiratory stimulants, such as doxapram. TASK-1 channels have also been suggested to play a role in circumventing cell apoptosis in a population of non-small-cell lung cancer cells. We propose that the inner vestibule of the TASK-1 channel, a known binding site of known TASK-1 inhibitors, BAY10000493 and BAY2341237, can be exploited via virtual screening to find other novel TASK-1 inhibitors. Our results show that by targeting the inner vestibule site, we found an active TASK-1 inhibitor. We suspect that this region of interest can be further exploited to discover additional TASK-1 inhibitors. Our initial success lends validity to our virtual screening methodology and parameters. In this study, we identified a novel TASK-1 inhibitor, KU124, which we verified using an in vitro assay.