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Summary: In angiosperms, wound‐derived signals travel through the vasculature to systemically activate defence responses throughout the plant. In Arabidopsis thaliana, activity of vasculature‐specific Clade 3 glutamate receptor‐like (GLR) channels is required for the transmission of electrical signals and cytosolic Ca2+ ([Ca2+]cyt) waves from wounded leaves to distal tissues, triggering activation of oxylipin‐dependent defences.Whether nonvascular plants mount systemic responses upon wounding remains unknown. To explore the evolution of systemic defence responses, we investigated electrical and calcium signalling in the nonvascular plant Marchantia polymorpha.We found that electrical signals and [Ca2+]cyt waves are generated in response to mechanical wounding and propagated to nondamaged distal tissues in M. polymorpha. Functional analysis of MpGLR, the only GLR encoded in the genome of M. polymorpha, indicates that its activity is necessary for the systemic transmission of wound‐induced electrical signals and [Ca2+]cyt waves, similar to vascular plants. However, spread of these signals is neither coupled to systemic accumulation of oxylipins nor to a transcriptional defence response in the distal tissues of wounded M. polymorpha plants.Our results suggest that lack of vasculature prevents translocation of additional signalling factors that, together with electrical signals and [Ca2+]cyt waves, contribute to systemic activation of defences in tracheophytes. See also the Commentary on this article by Beraldo & Alboresi, 244: 743–745. [ABSTRACT FROM AUTHOR]

Psychiatric disorders such as Bipolar disorder, Anxiety, Major depressive disorder, Schizophrenia, Attention-deficit/hyperactivity disorder, as well as neurological disorders such as Migraine, are linked by the evidence of altered calcium homeostasis. The disturbance of intra-cellular calcium homeostasis disrupts the activity of numerous ion channels including transient receptor potential (TRP) channels. TRP channel families comprise non-selective calcium-permeable channels that have been implicated in variety of physiological processes in the brain, as well as in the pathogenesis of psychiatric disorders. Through a comprehensive review of current research and experimentation, this investigation elucidates the role of TRP channels in psychiatric disorders. Furthermore, this review discusses about the exploration of epigenetics and TRP channels in psychiatric disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose: The single layer of cells lining all blood vessels, the endothelium, is a sophisticated signal co‐ordination centre that controls a wide range of vascular functions including the regulation of blood pressure and blood flow. To co‐ordinate activities, communication among cells is required for tissue level responses to emerge. While a significant form of communication occurs by the propagation of signals between cells, the mechanism of propagation in the intact endothelium is unresolved. Experimental Approach: Precision signal generation and targeted cellular manipulation was used in conjunction with high spatiotemporal mesoscale Ca2+ imaging in the endothelium of intact blood vessels. Key Results: Multiple mechanisms maintain communication so that Ca2+ wave propagation occurs irrespective of the status of connectivity among cells. Between adjoining cells, regenerative IP3‐induced IP3 production transmits Ca2+ signals and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. The inositide is itself sufficient to evoke regenerative phospholipase C‐dependent Ca2+ waves across coupled cells. None of gap junctions, Ca2+ diffusion or the release of extracellular messengers is required to support this type of intercellular Ca2+ signalling. In contrast, when discontinuities exist between cells, ATP released as a diffusible extracellular messenger transmits Ca2+ signals across the discontinuity and drives propagated vasodilation. Conclusion and Implications: These results show that signalling switches underlie endothelial cell‐to‐cell signal transmission and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding wave propagation and cell signalling in the endothelium. [ABSTRACT FROM AUTHOR]

Background and Purpose: Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease‐activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. Experimental Approach: The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N‐terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC–MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. Key Results: We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin‐10 (IL‐10) up‐regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. Conclusion and Implications: Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis. [ABSTRACT FROM AUTHOR]

Aims Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear. Methods and results We employed a large animal model, the female landrace pig, and used multiple in vivo and ex vivo approaches including pharmacological challenges, electrophysiology, and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta, or combined alpha-beta blockade), ganglionic blockade (hexamethonium), nor inhibition of hyperpolarization-activated cyclic nucleotide–gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened the action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate. Conclusion GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signalling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pacemaking. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in people treated with GLP-1 RAs. [ABSTRACT FROM AUTHOR]

Bitter taste receptors (TAS2Rs) expressed in extraoral tissues represent a whole-body sensory system, whose role and mechanisms could be of interest for the identification of new therapeutic targets. It is known that TAS2R46s in pre-contracted airway smooth muscle cells increase mitochondrial calcium uptake, leading to bronchodilation, and that several SNPs have been identified in its gene sequence. There are very few reports on the structure–function analysis of TAS2Rs. Thus, we delved into the subject by using mutagenesis and in silico studies. We generated a cellular model that expresses native TAS2R46 to evaluate the influence of the four most common SNPs on calcium fluxes following the activation of the receptor by its specific ligand absinthin. Then, docking studies were conducted to correlate the calcium flux results to the structural mutation. The analysed SNPs differently modulate the TAS2R46 signal cascade according to the altered protein domain. In particular, the SNP in the sixth transmembrane domain of the receptors did not modulate calcium homeostasis, while the SNPs in the sequence coding for the fourth transmembrane domain completely abolished the mitochondrial calcium uptake. In conclusion, these results indicate the fourth transmembrane domain of TAS2R46 is critical for the intrinsic receptor activity. [ABSTRACT FROM AUTHOR]

Yes-associated protein (YAP) and its homologue TAZ are transducers of several biochemical and biomechanical signals, integrating multiplexed inputs from the microenvironment into higher level cellular functions such as proliferation, differentiation and migration. Emerging evidence suggests that Ca2+ is a key second messenger that connects microenvironmental input signals and YAP/TAZ regulation. However, studies that directly modulate Ca2+ have reported contradictory YAP/TAZ responses: in some studies, a reduction in Ca2+ influx increases the activity of YAP/TAZ, while in others, an increase in Ca2+ influx activates YAP/TAZ. Importantly, Ca2+ and YAP/TAZ exhibit distinct spatiotemporal dynamics, making it difficult to unravel their connections from a purely experimental approach. In this study, we developed a network model of Ca2+ -mediated YAP/TAZ signalling to investigate how temporal dynamics and crosstalk of signalling pathways interacting with Ca2+ can alter the YAP/TAZ response, as observed in experiments. By including six signalling modules (e.g. GPCR, IP3-Ca2+ , kinases, RhoA, F-actin and Hippo-YAP/TAZ) that interact with Ca2+ , we investigated both transient and steady-state cell response to angiotensin II and thapsigargin stimuli. The model predicts that stimuli, Ca2+ transients and frequency-dependent relationships between Ca2+ and YAP/TAZ are primarily mediated by cPKC, DAG, CaMKII and F-actin. Simulation results illustrate the role of Ca2+ dynamics and CaMKII bistable response in switching the direction of changes in Ca2+ -induced YAP/TAZ activity. A frequency-dependent YAP/TAZ response revealed the competition between upstream regulators of LATS1/2, leading to the YAP/TAZ non-monotonic response to periodic GPCR stimulation. This study provides new insights into underlying mechanisms responsible for the controversial Ca2+ -YAP/TAZ relationship observed in experiments. KEY POINTS: YAP/TAZ integrates biochemical and biomechanical inputs to regulate cellular functions, and Ca2+ acts as a key second messenger linking cellular inputs to YAP/TAZ. Studies have reported contradictory Ca2+ -YAP/TAZ relationships for different cell types and stimuli. A network model of Ca2+ -mediated YAP/TAZ signalling was developed to investigate the underlying mechanisms of divergent Ca2+ -YAP/TAZ relationships. The model predicts context-dependent Ca2+ transient, CaMKII bistable response and frequency-dependent activation of LATS1/2 upstream regulators as mechanisms governing the Ca2+ -YAP/TAZ relationship. This study provides new insights into the underlying mechanisms of the controversial Ca2+ -YAP/TAZ relationship to better understand the dynamics of cellular functions controlled by YAP/TAZ activity.

This article is a Commentary on Giovannetti et al., 241: 1393–1400. [ABSTRACT FROM AUTHOR]

Diatoms are important primary producers in marine and freshwater environments, but little is known about the signalling mechanisms they use to detect changes in their environment. All eukaryotic organisms use Ca2+ signalling to perceive and respond to environmental stimuli, employing a range of Ca2+-permeable ion channels to facilitate the movement of Ca2+ across cellular membranes. We investigated the distribution of different families of Ca2+ channels in diatom genomes, with comparison to other members of the stramenopile lineage. The four-domain voltage-gated Ca2+ channels (Cav) are present in some centric diatoms but almost completely absent in pennate diatoms, whereas single-domain voltage-gated EukCatA channels were found in all diatoms. Glutamate receptors (GLRs) and pentameric ligand-gated ion channels (pLGICs) also appear to have been lost in several pennate species. Transient receptor potential (TRP) channels are present in all diatoms, but have not undergone the significant expansion seen in brown algae. All diatom species analysed lacked the mitochondrial uniporter (MCU), a highly conserved channel type found in many eukaryotes, including several stramenopile lineages. These results highlight the unique Ca2+-signalling toolkit of diatoms and indicate that evolutionary gains or losses of different Ca2+ channels may contribute to differences in cellular-signalling mechanisms between species. [ABSTRACT FROM AUTHOR]

Zinc is essential for many physiological functions, with a major role in digestive system, skin health, and learning and memory. On the cellular level, zinc is involved in cell proliferation and cell death. A selective zinc sensing receptor, ZnR/GPR39 is a Gq‐coupled receptor that acts via the inositol trisphosphate pathway to release intracellular Ca2+. The ZnR/GPR39 serves as a mediator between extracellular changes in Zn2+ concentration and cellular Ca2+ signalling. This signalling pathway regulates ion transporters activity and thereby controls the formation of transepithelial gradients or neuronal membrane potential, which play a fundamental role in the physiological function of these tissues. This review focuses on the role of Ca2+ signalling, and specifically ZnR/GPR39, with respect to the regulation of the Na+/H+ exchanger, NHE1, and of the K+/Cl− cotransporters, KCC1‐3, and also describes the physiological implications of this regulation. [ABSTRACT FROM AUTHOR]

Store operated Ca2+ entry (SOCE) is a ubiquitous signalling module with established roles in the immune system, secretion and muscle development. Recent evidence supports a complex role for SOCE in the nervous system. In this review we present an update of the current knowledge on SOCE function in the brain with a focus on its role as a regulator of brain activity and excitability [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling. [ABSTRACT FROM AUTHOR]

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG—EPG(B.g.)—the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Abstract Background Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars. Results The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs’ protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids. Conclusions We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells. Graphical Abstract

Background: Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars. Results: The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs' protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids. Conclusions: We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells. [ABSTRACT FROM AUTHOR]

Summary: Chitin oligomers (COs) are among the most common and active fungal elicitors of plant responses. Short‐chain COs from symbiotic arbuscular mycorrhizal fungi activate accommodation responses in the host root, while long‐chain COs from pathogenic fungi are acknowledged to trigger defence responses. The modulation of intracellular calcium concentration – a common second messenger in a wide variety of plant signal transduction processes – plays a central role in both signalling pathways with distinct signature features. Nevertheless, mounting evidence suggests that plant immunity and symbiosis signalling partially overlap at multiple levels. Here, we elaborate on recent findings on this topic, highlighting the nonbinary nature of chitin‐based fungal signals, their perception and their interpretation through Ca2+‐mediated intracellular signals. Based on this, we propose that plant perception of symbiotic and pathogenic fungi is less clear‐cut than previously described and involves a more complex scenario in which partially overlapping and blurred signalling mechanisms act upstream of the unambiguous regulation of gene expression driving accommodation or defence responses. [ABSTRACT FROM AUTHOR]

The stimulation of growth and development of crops using ionising radiation (radiation hormesis) has been reported by many research groups. However, specific genes contributing to the radiation stimulation of plant growth are largely unknown. In this work, we studied the impact of the low-dose γ-irradiation of barley seeds on the growth dynamics and gene expression of eight barley cultivars in a greenhouse experiment. Our findings confirmed that candidate genes of the radiation growth stimulation, previously established in barley seedlings (PM19L-like, CML31-like, and AOS2-like), are significant in radiation hormesis throughout ontogeny. In γ-stimulated cultivars, the expression of these genes was aligned with the growth dynamics, yield parameters, and physiological conditions of plants. We identified contrasting cultivars for future gene editing and found that the γ-stimulated cultivar possessed some specific abiotic stress-responsive elements in the promotors of candidate genes, possibly revealing a new level of radiation hormesis effect execution. These results can be used in creating new productive barley cultivars, ecological toxicology of radionuclides, and eustress biology studies. [ABSTRACT FROM AUTHOR]

An increase in intracellular [Ca2+] in exocrine acinar cells resident in the salivary glands or pancreas is a fundamental event that drives fluid secretion and exocytosis of proteins. Stimulation with secretagogues initiates Ca2+ signals with precise spatiotemporal properties thought to be important for driving physiological output. Both in vitro, in acutely isolated acini, and in vivo, in animals expressing genetically encoded indicators, individual cells appear specialized to initiate Ca2+ signals upon stimulation. Furthermore, these signals appear to spread to neighbouring cells. These properties are present in the absence of a conventional pacemaker mechanism dependent on the cyclical activation of Ca2+‐dependent or Ca2+‐conducting plasma membrane ion channels. In this article, we propose a model for ‘pacing’ intracellular Ca2+ signals in acinar cells based on the enhanced sensitivity of a subpopulation of individual cells and the intercellular diffusion through gap junctions of inositol 1,4,5‐trisphosphate and Ca2+ to neighbouring cells. [ABSTRACT FROM AUTHOR]

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8–5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark. [ABSTRACT FROM AUTHOR]

A fundamental function of an organ is the ability to perceive mechanical cues. Yet, how this is accomplished is not fully understood, particularly in plant roots. In plants, the majority of studies dealing with the effects of mechanical stress have investigated the aerial parts. However, in natural conditions roots are also subjected to mechanical cues, for example when the root encounters a hard obstacle during its growth or when the soil settles. To investigate root cellular responses to root compression, we developed a microfluidic system associated with a microvalve allowing the delivery of controlled and reproducible mechanical stimulations to the root. In this study, examining plants expressing the R-GECO1-mTurquoise calcium reporter, we addressed the root cell deformation and calcium increase induced by the mechanical stimulation. Lateral pressure applied on the root induced a moderate elastic deformation of root cortical cells and elicited a multicomponent calcium signal at the onset of the pressure pulse, followed by a second one at the release of the pressure. This indicates that straining rather than stressing of tissues is relevant to trigger the calcium signal. Although the intensity of the calcium response increases with the pressure applied, successive pressure stimuli led to a remarkable attenuation of the calcium signal. The calcium elevation was restricted to the tissue under pressure and did not propagate. Strain sensing, spatial restriction and habituation to repetitive stimulation represent the fundamental properties of root signalling in response to local mechanical stimulation. These data linking mechanical properties of root cells to calcium elevation contribute to elucidating the pathway allowing the root to adapt to the mechanical cues generated by the soil. [ABSTRACT FROM AUTHOR]

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane‐damaging pore‐forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under‐explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco‐2 intestinal epithelial cells and THP‐1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis‐like programmed cell death. TDH triggers caspase‐3 and 7 activations in the THP‐1 cells, while caspase‐7 activation is observed in the Caco‐2 cells. Interestingly, TDH appears to induce caspase‐independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP‐1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH‐induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase‐independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP‐1 cells, while PARP‐1 appears to play a critical role in the Caco‐2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non‐classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host–pathogen interaction processes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Raised serum concentrations of the sympathetic co‐transmitter neuropeptide Y (NPY) are linked to cardiovascular diseases. However, the signalling mechanism for vascular smooth muscle (VSM) constriction to NPY is poorly understood. Therefore, the present study investigated the mechanisms of NPY‐induced vasoconstriction in rat small mesenteric (RMA) and coronary (RCA) arteries. Experimental Approach: Third‐order mesenteric or intra‐septal arteries from male Wistar rats were assessed in wire myographs for isometric tension, VSM membrane potential and VSM intracellular Ca2+ events. Key Results: NPY stimulated concentration‐dependent vasoconstriction in both RMA and RCA, which was augmented by blocking NO synthase or endothelial denudation in RMA. NPY‐mediated vasoconstriction was blocked by the selective Y1 receptor antagonist BIBO 3304 and Y1 receptor protein expression was detected in both the VSM and endothelial cells in RMA and RCA. The selective Gβγ subunit inhibitor gallein and the PLC inhibitor U‐73122 attenuated NPY‐induced vasoconstriction. Signalling via the Gβγ–PLC pathway stimulated VSM Ca2+ waves and whole‐field synchronised Ca2+ flashes in RMA and increased the frequency of Ca2+ flashes in myogenically active RCA. Furthermore, in RMA, the Gβγ pathway linked NPY to VSM depolarization and generation of action potential‐like spikes associated with intense vasoconstriction. This depolarization activated L‐type voltage‐gated Ca2+ channels, as nifedipine abolished NPY‐mediated vasoconstriction. Conclusions and Implications: These data suggest that the Gβγ subunit, which dissociates upon Y1 receptor activation, initiates VSM membrane depolarization and Ca2+ mobilisation to cause vasoconstriction. This model may help explain the development of microvascular vasospasm during raised sympathetic nerve activity. [ABSTRACT FROM AUTHOR]

Bitter taste receptors (TAS2Rs) expressed in extraoral tissues represent a whole-body sensory system, whose role and mechanisms could be of interest for the identification of new therapeutic targets. It is known that TAS2R46s in pre-contracted airway smooth muscle cells increase mitochondrial calcium uptake, leading to bronchodilation, and that several SNPs have been identified in its gene sequence. There are very few reports on the structure–function analysis of TAS2Rs. Thus, we delved into the subject by using mutagenesis and in silico studies. We generated a cellular model that expresses native TAS2R46 to evaluate the influence of the four most common SNPs on calcium fluxes following the activation of the receptor by its specific ligand absinthin. Then, docking studies were conducted to correlate the calcium flux results to the structural mutation. The analysed SNPs differently modulate the TAS2R46 signal cascade according to the altered protein domain. In particular, the SNP in the sixth transmembrane domain of the receptors did not modulate calcium homeostasis, while the SNPs in the sequence coding for the fourth transmembrane domain completely abolished the mitochondrial calcium uptake. In conclusion, these results indicate the fourth transmembrane domain of TAS2R46 is critical for the intrinsic receptor activity.

Prostate cancer (PCa) is the primary cause of cancer-related deaths among men, and its incidence increases with age. Despite the availability of treatments, the high costs and complications associated with current cancer therapies pose challenges. Therefore, there is an urgent need to explore globally accessible and effective alternative medications to support prostate cancer treatment. Bioinformatics and system biology approaches were utilized to investigate repurposed medicines for prostate cancer treatment guided by host genomic biomarkers. The investigation identified 120 common differentially expressed genes (DEGs) and 24 hub genes (HubGs) as potential therapeutic targets by analyzing gene expression patterns and protein-protein interaction (PPI) networks. The top 10 hub genes CDK1, CCNA2, RRM2, ASPM, MELK, HJURP, DLGAP5, NCAPG, TTK, and HMMR are associated with the development and progression of prostate cancer. The DEGs enrichment analysis revealed significant axon guidance, cell-cell adhesion, extracellular space, and calcium signaling pathways associated with prostate cancer. Gene regulatory network analysis revealed transcription factors (FOXC1, GATA2 and E2F1 etc.) and micro-RNAs (hsa-mir-16–5p, hsa-mir-24–3p and hsa-mir-34a-5p etc.) as regulators of hub genes. The potential drug candidates, includingindirubin-3′-Monoxime (−7.7 kcal/mol), benzene sulfonamide (−8.8 kcal/mol), and cladribine (−6.6 kcal/mol) have been identified as the top 3 candidate drugs against CDK1, CCNA2 and RRM2, respectively. Besides, these drugs exhibit strong binding affinity with the target proteins and suggest their potential for repurposing in the treatment of prostate cancer. Collectively, the findings present valuable insights for advancing prostate cancer research, potentially leading to a deeper understanding and novel treatment strategies for relevant diseases.

Aims/hypothesis: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. Methods: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. Results: βS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. Conclusions/interpretation: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. Data availability: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498). [ABSTRACT FROM AUTHOR]

• Cases of unexplained pancreatitis in patients with gain-of-function RYR1 mutations. • RyRs are critically implicated in the pathophysiology of acute pancreatitis. • RYR1 mutations may be a risk factor for acute pancreatitis. • RyR inhibition may be an effective treatment for most forms of pancreatitis. Mutations in RYR1 encoding the ryanodine receptor (RyR) skeletal muscle isoform (RyR1) are a common cause of inherited neuromuscular disorders. Despite its expression in a wide range of tissues, non-skeletal muscle manifestations associated with RYR1 mutations have only been rarely reported. Here, we report three patients with a diagnosis of Central Core Disease (CCD), King-Denborough Syndrome (KDS) and Malignant Hyperthermia Susceptibility (MHS), respectively, who in addition to their (putative) RYR1 -related disorder also developed symptoms and signs of acute pancreatitis. In two patients, episodes were recurrent, with severe multisystem involvement and sequelae. RyR1-mediated calcium signalling plays an important role in normal pancreatic function but has also been critically implicated in the pathophysiology of acute pancreatitis, particularly in bile acid- and ethanol-induced forms. Findings from relevant animal models indicate that pancreatic damage in these conditions may be ameliorated through administration of the specific RyR1 antagonist dantrolene and other compounds modifying pancreatic metabolism including calcium signalling. These observations suggest that patients with RYR1 gain-of-function variants may be at increased risk of developing acute pancreatitis, a condition which should therefore be considered in the health surveillance of such individuals. [ABSTRACT FROM AUTHOR]

Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non‐sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age‐related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age‐related changes in the expression and function of metabotropic purinergic receptors (P2Y1, P2Y2 and P2Y4) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic‐mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month‐old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y2 and P2Y4 receptors and display purinergic‐induced Ca2+ responses that mimic those observed during pre‐hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. Key points: Age‐related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech.We investigated age‐related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non‐sensory supporting cells of mice displaying early‐onset (C57BL/6N) and late‐onset (C3H/HeJ) hearing loss.The expression of P2Y1, P2Y2 and P2Y4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y2 and P2Y4 was upregulated in the aged cochlea.P2Y2 and P2Y4 receptors were primarily responsible for the ATP‐induced Ca2+ responses in the supporting cells.The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains.We propose that the upregulation of purinergic‐mediated signalling in the aged cochlea is subsequent to age‐related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium. [ABSTRACT FROM AUTHOR]

Summary: All plants are electrically excitable, but only few are known to fire a well‐defined, all‐or‐nothing action potential (AP). The Venus flytrap Dionaea muscipula displays APs with an extraordinarily high firing frequency and speed, enabling the capture organ of this carnivorous plant to catch small animals as fast as flies. The number of APs triggered by the prey is counted and serves as the basis for decisions within the flytrap's hunting cycle. The archetypical Dionaea AP lasts 1 s and consists of five phases: Starting from the resting state, an initial cytosolic Ca2+ transient is followed by depolarization, repolarization and a transient hyperpolarization (overshoot) before the original membrane potential is finally recovered. When the flytrap matures and becomes excitable, a distinct set of ion channels, pumps and carriers is expressed, each mastering a distinct AP phase. [ABSTRACT FROM AUTHOR]

T lymphocytes (T cells) are an important sub-group of cells in our immune system responsible for cell-mediated adaptive responses and maintaining immune homeostasis. Abnormalities in T cell function, lead the way to the persistence of infection, impaired immunosurveillance, lack of suppression of cancer growth, and autoimmune diseases. Ion channels play a critical role in the regulation of T cell signaling and cellular function and are often overlooked and understudied. Little is known about the ion "channelome" and the interaction of ion channels in immune cells. This review aims to summarize the published data on the impact of ion channels on T cell function and disease. The importance of ion channels in health and disease plus the fact they are easily accessible by virtue of being expressed on the surface of plasma membranes makes them excellent drug targets. [ABSTRACT FROM AUTHOR]

Background & Aims: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium‐permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. Methods: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non‐disruptive tag in native PIEZO1 or endothelial‐specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. Results: PIEZO1 activation leads to nitric oxide synthase‐ and endothelium‐dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium‐dependent but independent of nitric oxide synthase. The TRPV4‐mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2, suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo‐osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. Conclusions: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures. [ABSTRACT FROM AUTHOR]

Diatoms are important primary producers in marine and freshwater environments, but little is known about the signalling mechanisms they use to detect changes in their environment. All eukaryotic organisms use Ca2+ signalling to perceive and respond to environmental stimuli, employing a range of Ca2+-permeable ion channels to facilitate the movement of Ca2+ across cellular membranes. We investigated the distribution of different families of Ca2+ channels in diatom genomes, with comparison to other members of the stramenopile lineage. The four-domain voltage-gated Ca2+ channels (Cav) are present in some centric diatoms but almost completely absent in pennate diatoms, whereas single-domain voltage-gated EukCatA channels were found in all diatoms. Glutamate receptors (GLRs) and pentameric ligand-gated ion channels (pLGICs) also appear to have been lost in several pennate species. Transient receptor potential (TRP) channels are present in all diatoms, but have not undergone the significant expansion seen in brown algae. All diatom species analysed lacked the mitochondrial uniporter (MCU), a highly conserved channel type found in many eukaryotes, including several stramenopile lineages. These results highlight the unique Ca2+-signalling toolkit of diatoms and indicate that evolutionary gains or losses of different Ca2+ channels may contribute to differences in cellular-signalling mechanisms between species.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

In response to Ca2+ signals, the evolutionarily‐conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM‐like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two‐hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin‐binding transcriptional activators (CAMTAs) and myosins, all of which possess tandem isoleucine‐glutamine (IQ) structural domains. Using the split‐luciferase complementation assay in planta and the yeast 2‐hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non‐IQ, CaM‐binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic‐nucleotide gated channel‐20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ‐protein and found that only CaM, CML13 and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13 and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+. Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein‐tagged versions of CaM, CML13 and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co‐expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins. Summary Statement: Plants possess many calmodulin‐like proteins (CMLs), but very few downstream targets of CMLs have been identified. We show that Arabidopsis CML13 and CML14 interact with proteins with multiple IQ‐domain proteins, including CAMTAs, myosins and IQDs, suggesting broad cellular roles for these CMLs. [ABSTRACT FROM AUTHOR]

Using a zebrafish ionocyte model, transcriptomics and genetic analyses were performed to identify pathways and genes involved in cell quiescence‐proliferation regulation. Gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses revealed that genes involved in transcription regulation, cell cycle, Foxo signalling and Wnt signalling pathway are enriched among the up‐regulated genes while those involved in ion transport, cell adhesion and oxidation–reduction are enriched among the down‐regulated genes. Among the top up‐regulated genes is FK506‐binding protein 5 (Fkbp5). Genetic deletion and pharmacological inhibition of Fkbp5 abolished ionocyte reactivation and impaired Akt signalling. Forced expression of a constitutively active form of Akt rescued the defects caused by Fkbp5 inhibition. These results uncover a key role of Fbkp5 in regulating the quiescence‐proliferation decision via Akt signalling. [ABSTRACT FROM AUTHOR]

Smooth muscle voltage‐gated K+ (Kv) channels in resistance arteries control vascular tone and contribute to the coupling of blood flow with local metabolic activity. Members of the Kv1 family are expressed in vascular smooth muscle and are modulated upon physiological elevation of local metabolites, including the glycolytic end‐product l‐lactate and superoxide‐derived hydrogen peroxide (H2O2). Here, we show that l‐lactate elicits vasodilatation of small‐diameter mesenteric arteries in a mechanism that requires lactate dehydrogenase (LDH). Using the inside‐out configuration of the patch clamp technique, we show that increases in NADH that reflect LDH‐mediated conversion of l‐lactate to pyruvate directly stimulate the activity of single Kv1 channels and significantly enhance the sensitivity of Kv1 activity to H2O2. Consistent with these findings, H2O2‐evoked vasodilatation was significantly greater in the presence of 10 mM l‐lactate relative to lactate‐free conditions, yet was abolished in the presence of 10 mM pyruvate, which shifts the LDH reaction towards the generation of NAD+. Moreover, the enhancement of H2O2‐induced vasodilatation was abolished in arteries from double transgenic mice with selective overexpression of the intracellular Kvβ1.1 subunit in smooth muscle cells. Together, our results indicate that the Kvβ complex of native vascular Kv1 channels serves as a nodal effector for multiple redox signals to precisely control channel activity and vascular tone in the face of dynamic tissue‐derived metabolic cues. Key points: Vasodilatation of mesenteric arteries by elevated external l‐lactate requires its conversion by lactate dehydrogenase.Application of either NADH or H2O2 potentiates single Kv channel currents in excised membrane patches from mesenteric artery smooth muscle cells.The binding of NADH enhances the stimulatory effects of H2O2 on single Kv channel activity.The vasodilatory response to H2O2 is differentially modified upon elevation of external l‐lactate or pyruvate.The presence of l‐lactate enhances the vasodilatory response to H2O2 via the Kvβ subunit complex in smooth muscle. [ABSTRACT FROM AUTHOR]

ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14–19). Octanol had no effect on Ca2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca2+ pump expressions and in IP3–Ca2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP3-mediated Ca2+ signalling inhibition by CBX cannot be excluded. [ABSTRACT FROM AUTHOR]

Most patients with malignant hyperthermia susceptibility diagnosed by the in vitro caffeine–halothane contracture test (CHCT) develop excessive force in response to halothane but not caffeine (halothane-hypersensitive). Hallmarks of halothane-hypersensitive patients include high incidence of musculoskeletal symptoms at rest and abnormal calcium events in muscle. By measuring sensitivity to halothane of myotubes and extending clinical observations and cell-level studies to a large group of patients, we reach new insights into the pathological mechanism of malignant hyperthermia susceptibility. Patients with malignant hyperthermia susceptibility were classified into subgroups HH and HS (positive to halothane only and positive to both caffeine and halothane). The effects on [Ca2+] cyto of halothane concentrations between 0.5 and 3 % were measured in myotubes and compared with CHCT responses of muscle. A clinical index that summarises patient symptoms was determined for 67 patients, together with a calcium index summarising resting [Ca2+] cyto and spontaneous and electrically evoked Ca2+ events in their primary myotubes. Halothane-hypersensitive myotubes showed a higher response to halothane 0.5% than the caffeine-halothane hypersensitive myotubes (P <0.001), but a lower response to higher concentrations, comparable with that used in the CHCT (P =0.055). The HH group had a higher calcium index (P <0.001), but their clinical index was not significantly elevated vs the HS. Principal component analysis identified electrically evoked Ca2+ spikes and resting [Ca2+] cyto as the strongest variables for separation of subgroups. Enhanced sensitivity to depolarisation and to halothane appear to be the primary, mutually reinforcing and phenotype-defining defects of halothane-hypersensitive patients with malignant hyperthermia susceptibility. [ABSTRACT FROM AUTHOR]