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Two-pore channels are pathophysiologically important Na + - and Ca 2+ -permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P 2 , they are more Na + -selective than when gated by the Ca 2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P 2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na + permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na + to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na + over Ca 2+ . Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions., Competing Interests: Competing interests statement:The authors declare no competing interest.

Aims: Our recent study demonstrated that increased Ca2+ sparks and spontaneous transient outward currents (STOCs) played an important role in uterine vascular tone and haemodynamic adaptation to pregnancy. The present study examined the role of ryanodine receptor (RyR) subtypes in regulating Ca2+ sparks/STOCs and myogenic tone in uterine arterial adaptation to pregnancy., Methods and Results: Uterine arteries isolated from non-pregnant and near-term pregnant sheep were used in the present study. Pregnancy increased the association of α and β1 subunits of large-conductance Ca2+-activated K+ (BKCa) channels and enhanced the co-localization of RyR1 and RyR2 with the β1 subunit in the uterine artery. In contrast, RyR3 was not co-localized with BKCa β1 subunit. Knockdown of RyR1 or RyR2 in uterine arteries of pregnant sheep downregulated the β1 but not α subunit of the BKCa channel and decreased the association of α and β1 subunits. Unlike RyR1 and RyR2, knockdown of RyR3 had no significant effect on either expression or association of BKCa subunits. In addition, knockdown of RyR1 or RyR2 significantly decreased Ca2+ spark frequency, suppressed STOCs frequency and amplitude, and increased pressure-dependent myogenic tone in uterine arteries of pregnant animals. RyR3 knockdown did not affect Ca2+ sparks/STOCs and myogenic tone in the uterine artery., Conclusion: Together, the present study demonstrates a novel mechanistic paradigm of RyR subtypes in the regulation of Ca2+ sparks/STOCs and uterine vascular tone, providing new insights into the mechanisms underlying uterine vascular adaptation to pregnancy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Novel structures of the human TRPA1 channel were determined in the presence of the agonist iodoacetamide and the antagonist A-967079, to reveal the open and closed states of the channel, respectively. The structures further revealed the location of Ca 2+ modulatory site that is likely conserved among several TRP subgroups., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Conformational changes in amyloidogenic proteins, such as β-amyloid protein, prion proteins, and α-synuclein, play a critical role in the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease, prion disease, and Lewy body disease. The disease-associated proteins possess several common characteristics, including the ability to form amyloid oligomers with β-pleated sheet structure, as well as cytotoxicity, although they differ in amino acid sequence. Interestingly, these amyloidogenic proteins all possess the ability to bind trace metals, can regulate metal homeostasis, and are co-localized at the synapse, where metals are abundantly present. In this review, we discuss the physiological roles of these amyloidogenic proteins in metal homeostasis, and we propose hypothetical models of their pathogenetic role in the neurodegenerative process as the loss of normal metal regulatory functions of amyloidogenic proteins. Notably, these amyloidogenic proteins have the capacity to form Ca 2+ -permeable pores in membranes, suggestive of a toxic gain of function. Therefore, we focus on their potential role in the disruption of Ca 2+ homeostasis in amyloid-associated neurodegenerative diseases.

Ca 2+ is a universal second messenger in many signaling pathways in all eukaryotes including plants. Transient changes in [Ca 2+ ]cyt are rapidly generated upon a diverse range of stimuli such as drought, heat, wounding, and biotic stresses (infection by pathogenic and symbiotic microorganisms), as well as developmental cues. It has been known for a while that [Ca 2+ ]cyt transient signals play crucial roles to activate plant immunity and recently significant progresses have been made in this research field. However the identity and regulation of ion channels that are involved in defense related Ca 2+ signals are still enigmatic. Members of two ligand gated ion channel families, glutamate receptor-like channels (GLRs) and cyclic nucleotide-gated channels (CNGCs) have been implicated in immune responses; nevertheless more precise data to understand their direct involvement in the creation of Ca 2+ signals during immune responses is necessary. Furthermore, the study of other ion channel groups is also required to understand the whole picture of the intra- and inter-cellular Ca 2+ signalling network. In this review we summarize Ca 2+ signals in plant immunity from an ion channel point of view and discuss future challenges in this exciting research field., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ca 2+ flux across the inner mitochondrial membrane (IMM) regulates cellular bioenergetics, intra-cellular cytoplasmic Ca 2+ signals, and various cell death pathways. Ca 2+ entry into the mitochondria occurs due to the highly negative membrane potential (ΔΨ m ) through a selective inward rectifying MCU channel. In addition to being regulated by various mitochondrial matrix resident proteins such as MICUs, MCUb, MCUR1 and EMRE, the channel is transcriptionally regulated by upstream Ca 2+ cascade, post transnational modification and by divalent cations. The mode of regulation either inhibits or enhances MCU channel activity and thus regulates mitochondrial metabolism and cell fate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The dehydration of ion-water complexes prior to ion channel transit has focused on channel protein-mediated dissociation of water. Ion dehydration by the membrane electric field has not previously been considered. Near membrane electric fields have previously been shown to cause the disassociation of non-covalently bound small molecule-small molecule, small molecule-protein, and protein-protein complexes. It is well known that cosmotropic, structure making ions such as calcium and sodium significantly bind multiple water ions in solution. It is also known that these ions are often not hydrated as they pass through membrane ion channels. Using capillary electrophoresis, the range of electric fields needed to strip water molecules from calcium ions has been measured. Ion migration velocity is a linear function of the electric field. At low electric fields, the migration rate of calcium ion was shown to be linearly related to the applied electric field. Using a form of the Stoke's equation applicable to ion migration, the hydrated calcium radius was found to be 0.334nm, corresponding to a water hydration shell of 5.09 water molecules. At higher electric fields, the slope of the calcium migration velocity as a function of the electric field increased, which was modeled as a decrease in the radius of the migrating ion as the water was removed. Using a tanh function to model the transition of the ion from a hydrated to a stripped state, the transition had a midpoint at 446V/cm, and was 88% complete at 587V/cm with a correlation coefficient of 0.9996. The migration velocity of the stripped calcium ion was found to be a function of both the decrease in radius and an increase in the effective, electronic viscosity of the dipole medium through which the dehydrated ion moved. The size of the electric field needed to dehydrate calcium occurs 6-7nm from the cell membrane. Calcium ions within this distance from the membrane will be devoid of water molecules when they reach the calcium selective channel pore entrances, all known to be approximately 1-2nm from the membrane. No matter what the calcium pore structure, calcium ions reaching the channel entrance will be devoid of a water shell., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Calcium, phosphate, and magnesium are multivalent cations that are important for many biologic and cellular functions. The kidneys play a central role in the homeostasis of these ions. Gastrointestinal absorption is balanced by renal excretion. When body stores of these ions decline significantly, gastrointestinal absorption, bone resorption, and renal tubular reabsorption increase to normalize their levels. Renal regulation of these ions occurs through glomerular filtration and tubular reabsorption and/or secretion and is therefore an important determinant of plasma ion concentration. Under physiologic conditions, the whole body balance of calcium, phosphate, and magnesium is maintained by fine adjustments of urinary excretion to equal the net intake. This review discusses how calcium, phosphate, and magnesium are handled by the kidneys., (Copyright © 2015 by the American Society of Nephrology.)

Sound encoding is mediated by Ca(2+) influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca(2+) current through CaV1.3 channels and Ca(2+) homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca(2+) imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca(2+) influx in inner hair cells via fueling Ca(2+)-ATPases to avoid an increase in cytosolic [Ca(2+)] and subsequent Ca(2+)/calmodulin-dependent inactivation of CaV1.3 channels., (Copyright © 2014 the authors 0270-6474/14/346843-06$15.00/0.)

Calcium ions function as a key second messenger ion in eukaryotes. Spatially and temporally defined cytoplasmic Ca2+ signals are shaped through the concerted activity of ion channels, exchangers, and pumps in response to diverse stimuli; these signals are then decoded through the activity of Ca2+‐binding sensor proteins. In plants, Ca2+ signaling is central to both pattern‐ and effector‐triggered immunity, with the generation of characteristic cytoplasmic Ca2+ elevations in response to potential pathogens being common to both. However, despite their importance, and a long history of scientific interest, the transport proteins that shape Ca2+ signals and their integration remain poorly characterized. Here, we discuss recent work that has both shed light on and deepened the mysteries of Ca2+ signaling in plant immunity. [ABSTRACT FROM AUTHOR]

Facilitation of cardiac function in response to signals from the sympathetic nervous system is initiated by the phosphorylation of L-type voltage-dependent Ca2+ channels (VDCCs) by protein kinase A (PKA), which in turn is activated by β-adrenoceptors. Among the five subunits (α1, β, α2/δ, and γ) of VDCCs, the α1 subunit and the family of β subunits are substrates for PKA-catalyzed phosphorylation; however, the subunit responsible for β-adrenergic augmentation of Ca2+ channel function has yet to be specifically identified. Here we show that the VDCC β2 subunit is required for PKA phosphorylation upon sympathetic acceleration. In mice with β2 subunit-null mutations, cardiac muscle contraction in response to isoproterenol was reduced and there was no significant increase in Ca2+ channel currents upon PKA-dependent phosphorylation. These findings indicate that within the sympathetic nervous system the β2 subunit of VDCCs is required for physiological PKA-dependent channel phosphorylation.

The Golgi apparatus is a membrane organelle located in the center of the protein processing and trafficking pathway. It consists of sub-compartments with distinct biochemical compositions and functions. Main functions of the Golgi, including membrane trafficking, protein glycosylation, and sorting, require a well-maintained stable microenvironment in the sub-compartments of the Golgi, along with metal ion homeostasis. Metal ions, such as Ca2+, Mn2+, Zn2+, and Cu2+, are important cofactors of many Golgi resident glycosylation enzymes. The homeostasis of metal ions in the secretory pathway, which is required for proper function and stress response of the Golgi, is tightly regulated and maintained by transporters. Mutations in the transporters cause human diseases. Here we provide a review specifically focusing on the transporters that maintain Golgi metal ion homeostasis under physiological conditions and their alterations in diseases.

Despite responses to initial treatment of photodynamic therapy (PDT) being promising, a recurrence rate exists. Thus, finding novel therapeutic targets to enhance PDT efficacy is an urgent need. Reports indicate that connexin (Cx) 40 plays an important role in tumor angiogenesis and growth. However, it is unknown whether Cx40-composed channels have effects on PDT efficacy. The study uniquely demonstrated that Cx40-formed channels could enhance the phototoxicity of PDT to malignant cells in vitro and in vivo. Specifically, Cx40-formed channels at high cell density could increase PDT photocytotoxicity. This action was substantially restricted when Cx40 expression was not induced or Cx40 channels were restrained. Additionally, the presence of Cx40-composed channels enhanced the phototoxicity of PDT in the tumor xenografts. The above results indicate that enhancing the function of Cx40-formed channels increases PDT efficacy. The enhancement of PDT efficacy mediated by Cx40 channels was related with intracellular pathways mediated by ROS and calcium pathways, but not the lipid peroxide-mediated pathway. This work demonstrates the capacity of Cx40-mediated channels to increase PDT efficacy and suggests that therapeutic strategies designed to maintain or enhance Cx40 expression and/or channels composed by Cx40 may increase the therapeutic efficacy of PDT.

Despite responses to initial treatment of photodynamic therapy (PDT) being promising, a recurrence rate exists. Thus, finding novel therapeutic targets to enhance PDT efficacy is an urgent need. Reports indicate that connexin (Cx) 40 plays an important role in tumor angiogenesis and growth. However, it is unknown whether Cx40-composed channels have effects on PDT efficacy. The study uniquely demonstrated that Cx40-formed channels could enhance the phototoxicity of PDT to malignant cells in vitro and in vivo. Specifically, Cx40-formed channels at high cell density could increase PDT photocytotoxicity. This action was substantially restricted when Cx40 expression was not induced or Cx40 channels were restrained. Additionally, the presence of Cx40-composed channels enhanced the phototoxicity of PDT in the tumor xenografts. The above results indicate that enhancing the function of Cx40-formed channels increases PDT efficacy. The enhancement of PDT efficacy mediated by Cx40 channels was related with intracellular pathways mediated by ROS and calcium pathways, but not the lipid peroxide-mediated pathway. This work demonstrates the capacity of Cx40-mediated channels to increase PDT efficacy and suggests that therapeutic strategies designed to maintain or enhance Cx40 expression and/or channels composed by Cx40 may increase the therapeutic efficacy of PDT. [ABSTRACT FROM AUTHOR]

Whether the mitochondrial permeability transition pore (PTP), also called mitochondrial megachannel (MMC), originates from the F‐ATP synthase is a matter of controversy. This hypothesis is supported both by site‐directed mutagenesis of specific residues of F‐ATP synthase affecting regulation of the PTP/MMC and by deletion of specific subunits causing dramatic changes in channel conductance. In contrast, human cells lacking an assembled F‐ATP synthase apparently display persistence of the PTP. We discuss recent data that shed new light on this controversy, supporting the conclusion that the PTP/MMC originates from a Ca2+‐dependent conformational change in F‐ATP synthase allowing its reversible transformation into a high‐conductance channel. [ABSTRACT FROM AUTHOR]

Conformational changes in amyloidogenic proteins, such as β-amyloid protein, prion proteins, and α-synuclein, play a critical role in the pathogenesis of numerous neurodegenerative diseases, including Alzheimer’s disease, prion disease, and Lewy body disease. The disease-associated proteins possess several common characteristics, including the ability to form amyloid oligomers with β-pleated sheet structure, as well as cytotoxicity, although they differ in amino acid sequence. Interestingly, these amyloidogenic proteins all possess the ability to bind trace metals, can regulate metal homeostasis, and are co-localized at the synapse, where metals are abundantly present. In this review, we discuss the physiological roles of these amyloidogenic proteins in metal homeostasis, and we propose hypothetical models of their pathogenetic role in the neurodegenerative process as the loss of normal metal regulatory functions of amyloidogenic proteins. Notably, these amyloidogenic proteins have the capacity to form Ca2+-permeable pores in membranes, suggestive of a toxic gain of function. Therefore, we focus on their potential role in the disruption of Ca2+ homeostasis in amyloid-associated neurodegenerative diseases.

The article offers information on the plasma membrane (PM) receptors for extracellular ATP (eATP) that function in such processes as pain perception and vasodilation. Topics discussed include effects of eATP on roots gravitropism, growth and development; role of eATP in increasing plant free Ca2+( cytosolic, nuclear, mitochondrial ); and the discovery of arabidopsis thaliana DORN1.

Calcium (Ca2+) signaling plays a major role in regulating multiple processes in living cells. The photoreceptor potential in Chlamydomonas triggers the generation of all or no flagellar Ca2+ currents that cause membrane depolarization across the eyespot and flagella. Modulation in membrane potential causes changes in the flagellar waveform, and hence, alters the beating patterns of Chlamydomonas flagella. The rhodopsin-mediated eyespot membrane potential is generated by the photoreceptor Ca2+ current or P-current however, the flagellar Ca2+ currents are mediated by unidentified voltage-gated calcium (VGCC or CaV) and potassium channels (VGKC). The voltage-gated ion channel that associates with ChRs to generate Ca2+ influx across the flagella and its cellular distribution has not yet been identified. Here, we identified putative VGCCs from algae and predicted their novel properties through insilico analysis. We further present experimental evidence on Chlamydomonas reinhardtii VGCCs to predict their novel physiological roles. Our experimental evidences showed that CrVGCC4 localizes to the eyespot and flagella of Chlamydomonas and associates with channelrhodopsins (ChRs). Further in silico interactome analysis of CrVGCCs suggested that they putatively interact with photoreceptor proteins, calcium signaling, and intraflagellar transport components. Expression analysis indicated that these VGCCs and their putative interactors can be perturbed by light stimuli. Collectively, our data suggest that VGCCs in general, and VGCC4 in particular, might be involved in the regulation of the photobehavioral response of Chlamydomonas. [ABSTRACT FROM AUTHOR]

Calcium-permeable channels underpin elevations of free calcium that encode specific signals in stress adaptation, development and immunity. Identifying the genes encoding these channels remains a central goal of plant signalling research. Evidence now suggests that members of the plant annexin family function as unconventional calcium-permeable channels, with roles in development and stress signalling. Arabidopsis annexin 1 mediates a plasma membrane calcium-permeable conductance in roots that is activated by reactive oxygen species. Recombinant annexin 1 forms a very similar conductance in planar lipid bilayers, indicating that this protein could facilitate the in vivo conductance directly. The annexin 1 mutant is impaired in salinity-induced calcium signalling. Protein–protein interactions, post-translational modification and dynamic association with membranes could all influence annexin-mediated calcium signalling and are reviewed here. The prospect of annexins playing roles in calcium signalling events in symbiosis and immunity are considered.

Mechanosensory feedback of the internal reproductive state drives decisions about when and where to reproduce. 1 For instance, stretch in the Drosophila reproductive tract produced by artificial distention or from accumulated eggs regulates the attraction to acetic acid to ensure optimal oviposition. 2 How such mechanosensory feedback modulates neural circuits to coordinate reproductive behaviors is incompletely understood. We previously identified a stretch-dependent homeostat that regulates egg laying in Caenorhabditis elegans. Sterilized animals lacking eggs show reduced Ca2+ transient activity in the presynaptic HSN command motoneurons that drive egg-laying behavior, while animals forced to accumulate extra eggs show dramatically increased circuit activity that restores egg laying. 3 Interestingly, genetic ablation or electrical silencing of the HSNs delays, but does not abolish, the onset of egg laying, 3,4,5 with animals recovering vulval muscle Ca2+ transient activity upon egg accumulation. 6 Using an acute gonad microinjection technique to mimic changes in pressure and stretch resulting from germline activity and egg accumulation, we find that injection rapidly stimulates Ca2+ activity in both neurons and muscles of the egg-laying circuit. Injection-induced vulval muscle Ca2+ activity requires L-type Ca2+ channels but is independent of presynaptic input. Conversely, injection-induced neural activity is disrupted in mutants lacking the vulval muscles, suggesting "bottom-up" feedback from muscles to neurons. Direct mechanical prodding activates the vulval muscles, suggesting that they are the proximal targets of the stretch-dependent stimulus. Our results show that egg-laying behavior in C. elegans is regulated by a stretch-dependent homeostat that scales postsynaptic muscle responses with egg accumulation in the uterus. [Display omitted] • Acute stretch drives vulval muscle and neuron activity of the egg-laying circuit • L-type calcium channels mediate the mechanosensory response • Feedback from muscles promotes a stretch-evoked response in the neurons • The muscles, not neurons, are important proximal targets of the stretch stimulus Feedback of internal states is essential for animals' behavioral decisions. Here, Medrano and Collins show that egg accumulation and feedback of internal stretch promotes egg-laying circuit activity and behavior in the nematode worm, C. elegans. This bottom-up signaling acts alongside top-down sensory feedback to optimize reproductive behaviors. [ABSTRACT FROM AUTHOR]

The Paramecium tetraurelia cell is highly organised, with regularly spaced elements pertinent to Ca 2+ signalling under epigenetic control. Vesicles serving as stationary Ca 2+ stores or undergoing trafficking contain Ca 2+ -release channels ( Pt CRCs) which, according to sequence and domain comparison, are related either to inositol 1,4,5-trisphosphate (InsP 3 ) receptors (IP 3 R) or to ryanodine receptor-like proteins (RyR-LP) or to both, with intermediate characteristics or deviation from conventional domain structure. Six groups of such Pt CRCs have been found. The ryanodine-InsP 3 -receptor homology (RIH) domain is not always recognisable, in contrast to the channel domain with six trans-membrane domains and the pore between transmembrane domain 5 and 6. Two CRC subtypes tested more closely, Pt CRC-II and Pt CRC-IV, with and without an InsP 3 -binding domain, reacted to InsP 3 and to caffeine, respectively, and hence represent IP 3 Rs and RyR-LPs. IP 3 Rs occur in the contractile vacuole complex where they allow for stochastic constitutive Ca 2+ reflux into the cytosol. RyR-LPs are localised to cortical Ca 2+ stores; they are engaged in dense core-secretory vesicle exocytosis by Ca 2+ release, superimposed by Ca 2+ -influx via non-ciliary Ca 2+ -channels. One or two different types of Pt CRCs also occur in other vesicles undergoing trafficking. Since the Pt CRCs described combine different features they are considered derivatives of primitive precursors. The highly regular, epigenetically controlled design of a Paramecium cell allows it to make Ca 2+ available very locally, in a most efficient way, along predetermined trafficking pathways, including regulation of exocytosis, endocytosis, phagocytosis and recycling phenomena. The activity of cilia is also regulated by Ca 2+ , yet independently from any CRCs, by de- and hyperpolarisation of the cell membrane potential. [ABSTRACT FROM AUTHOR]

The ciliated protozoan, Paramecium tetraurelia has a high basic Ca 2+ leakage rate which is counteracted mainly by export through a contractile vacuole complex, based on its V-type H + -ATPase activity. In addition Paramecium cells dispose of P-type Ca 2+ -ATPases, i.e. a plasmamembrane and a sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (PMCA, SERCA). Antiporter systems are to be expected, as inferred from indirect evidence. Among the best known cytosolic Ca 2+ -binding proteins, calmodulin activates Ca 2+ influx channels in the somatic cell membrane, but inactivates Ca 2+ influx channels in cilia, where it, thus, ends ciliary reversal induced by depolarization via channels in the somatic cell membrane. Centrin inactivates Ca 2+ signals after stimulation by its high capacity/low affinity binding sites, whereas its high affinity sites regulate some other functions. Cortical Ca 2+ stores (alveolar sacs) are activated during stimulated trichocyst exocytosis and thereby mediate store-operated Ca 2+ entry (SOCE). Ca 2+ release channels (CRCs) localised to alveoli and underlying SOCE are considered as Ryanodine receptor-like proteins (RyR-LPs) which are members of a CRC family with 6 subfamilies. These also encompass genuine inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and intermediates between the two channel types. All IP 3 R/RyR-type CRCs possess six carboxyterminal transmembrane domains (TMD), with a pore domain between TMD 5 and 6, endowed with a characteristic selectivity filter. There are reasons to assume a common ancestor molecule for such channels and diversification further on in evolution. The distinct distribution of specific CRCs in the different vesicles undergoing intracellular trafficking suggests constitutive formation of very locally restricted Ca 2+ signals during vesicle–vesicle interaction. In summary, essential steps of Ca 2+ signalling already occur at this level of evolution, including an unexpected multitude of CRCs. For dis-/similarities with other bikonts see “Conclusions”. [ABSTRACT FROM AUTHOR]

Molecular cascades of calcium homeostasis and signalling (Ca 2+ pumps, channels, cation exchangers, and Ca 2+ -binding proteins) emerged in prokaryotes and further developed at the unicellular stage of eukaryote evolution. With progressive evolution, mechanisms of signalling became diversified reflecting multiplication and specialisation of Ca 2+ -regulated cellular activities. Recent genomic analysis of organisms from different systematic positions, combined with proteomic and functional probing invigorated expansion in our understanding of the evolution of Ca 2+ signalling. Particularly impressive is the consistent role of Ca 2+ -ATPases/pumps, calmodulin and calcineurin from very early stages of eukaryotic evolution, although with interspecies differences. Deviations in Ca 2+ handling and signalling are observed between vertebrates and flowering plants as well as between protists at the basis of the two systematic categories, Unikonta (for example choanoflagellates) and Bikonta (for example ciliates). Only the B-subunit of calcineurin, for instance, is maintained to regulate highly diversified protein kinases for stress defence in flowering plants, whereas the complete dimeric protein, in vertebrates up to humans, regulates gene transcription, immune-defence and plasticity of the brain. Calmodulin is similarly maintained throughout evolution, but in plants a calmoldulin-like domain is integrated into protein kinase molecules. The eukaryotic cell has inherited and invented many mechanisms to exploit the advantages of signalling by Ca 2+ , and there is considerable overall similarity in basic processes of Ca 2+ regulation and signalling during evolution, although some details may vary. [ABSTRACT FROM AUTHOR]

Sound encoding is mediated by Ca2+ influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca2+ current through CaV1.3 channels and Ca2+ homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca2+ imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca2+ influx in inner hair cells via fueling Ca2+-ATPases to avoid an increase in cytosolic [Ca2+] and subsequent Ca2+/calmodulin-dependent inactivation of CaV1.3 channels. [ABSTRACT FROM AUTHOR]

Abstract: Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to Gq/11 proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca2+. The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP3 receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca2+ stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability. [Copyright &y& Elsevier]

Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4μM GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca2+ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma. [Copyright &y& Elsevier]

Abstract: The receptors for the second messenger InsP3 comprise a family of closely related ion channels that release Ca2+ from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP3Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca2+ signals, but the spatial organization of InsP3Rs in Ca2+ stores is poorly characterized. Using nuclei isolated from insect Sf9 cells and freeze-dry rotary shadowing, we have addressed this by directly visualizing the cytoplasmic domain of InsP3R located on the cytoplasmic side of the nuclear envelope. Identification of ∼15nm structures as the cytoplasmic domain of InsP3R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP3R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP3R antibody. Over-expression of InsP3R did not result in the formation of arrays or clusters with channels touching each other. Gold-labeling suggests that the channel amino terminus resides near the center of the cytoplasmic tetrameric quaternary structure. The combination of nuclear isolation with freeze-drying and rotary shadow techniques allows direct visualization of InsP3Rs in native nuclear envelopes and can be used to determine their spatial distribution and density. [ABSTRACT FROM AUTHOR]

Abstract: Annexins are soluble proteins that undergo conditional association or insertion into membranes. Plants contain several isoforms, each of which may be capable of supporting more than one in vitro activity such as actin binding, phosphodiesterase activity, peroxidase activity, and cation transport. Enzymatic activities are modulated by lipid binding, Ca2+ and S-glutathionylation. A given annexin can occupy diverse positions in cells, including the apoplast and organelles, with membrane association and expression often as a consequence of perception of a stimulus (for example, salinity, nodulation) that may involve reactive oxygen species. The ability to translocate Ca2+ in vitro identifies annexins as a novel class of plant ion transporters that could account for channel activities in plasma- and endo-membranes and suggests roles in plant signalling and development. Studies on loss of function or overexpressing lines firmly implicate annexins as participating in the regulation of drought and salinity stress responses. How annexins operate in vivo, in terms of localisation and protein function now needs to be determined. With several tiers of regulation (space, time, post-translational modification) potentially operating on the soluble and membrane populations, annexins are complex components of plant cell Ca2+ networks. [Copyright &y& Elsevier]

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: Lipid signaling and phosphorylation cascades are fundamental to calcium signaling networks. In this review, we will discuss the recent laboratory findings for the phospholipase A2 (PLA2)/protein kinase C (PKC) pathway within cellular calcium networks. The complexity and connectivity of these ubiquitous cellular signals make interpretation of experimental results extremely challenging. We present here computational methods which have been developed to conquer such complex data, and how they can be used to make models capable of accurately predicting cellular responses within multiple calcium signaling pathways. We propose that information obtained from network analysis and computational techniques provides a rich source of knowledge which can be directly translated to the laboratory benchtop. [Copyright &y& Elsevier]

The Golgi apparatus is a membrane organelle located in the center of the protein processing and trafficking pathway. It consists of sub-compartments with distinct biochemical compositions and functions. Main functions of the Golgi, including membrane trafficking, protein glycosylation, and sorting, require a well-maintained stable microenvironment in the sub-compartments of the Golgi, along with metal ion homeostasis. Metal ions, such as Ca2+, Mn2+, Zn2+, and Cu2+, are important cofactors of many Golgi resident glycosylation enzymes. The homeostasis of metal ions in the secretory pathway, which is required for proper function and stress response of the Golgi, is tightly regulated and maintained by transporters. Mutations in the transporters cause human diseases. Here we provide a review specifically focusing on the transporters that maintain Golgi metal ion homeostasis under physiological conditions and their alterations in diseases. [ABSTRACT FROM AUTHOR]

Abstract: Calcium modulates the 5-HT3 receptor response by reducing peak current amplitude and increasing rates of activation, deactivation and desensitisation, but the binding site(s) and mechanism(s) of this modulation are unknown. Here we study residues that may be involved in calcium binding in two partially overlapping regions of the extracellular domain (E213–E215–E218 and D204–E218–V219). The modulatory effects of calcium were assessed by radioligand binding and whole-cell patch-clamp. Comparisons of [3H]granisetron binding showed an increase in K d in 10mM calcium that was abolished by the substitutions E213Q, E215Q, D204N and V219L. E218Q mutant receptors displayed no specific binding or function, and immunofluorescence showed that they did not reach the cell surface. E213Q increased inherent rates of desensitisation, but the relative effects of calcium on these rates, and on the reduction in current amplitude, were similar to wild type receptors. Current responses and calcium-mediated effects at E215Q mutant receptors were indistinguishable from wild type. D204N and V219L mutants were non-functional. A calcium impermeable mutant (E277A/S297R) revealed no changes in peak amplitude or kinetics with increased calcium. Our results are consistent with residues D204, E218 and V219 participating in receptor assembly, structure and/or trafficking to the plasma membrane, and we speculate that this might rely upon the stabilising effect of bound calcium. E213, E215, D204 and V219 may contribute to a calcium binding site that is responsible for the calcium-mediated effects on ligand binding. However, the major site for calcium-dependent modulation of the 5-HT3 current is located within the ion channel or cell interior. [Copyright &y& Elsevier]