Antony Galione

Antony Galione
University of Oxford | OX · Department of Pharmacology

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252
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Introduction
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Publications

Publications (252)
Article
We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca(2+) release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2...
Article
Full-text available
Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic s...
Article
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In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocr...
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Background Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely un...
Article
Spontaneous Ca ²⁺ waves, also termed store-overload-induced Ca ²⁺ release (SOICR), in cardiac cells can trigger ventricular arrhythmias especially in failing hearts. SOICR occurs when Ryanodine receptors (RyRs) are activated by an increase in sarcoplasmic reticulum (SR) luminal Ca ²⁺. Carvedilol is one of the most effective drugs for preventing arr...
Article
Previously, we have reported that nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are calcium mobilising messengers that have significant roles in excitation-contraction coupling and disease development of the heart. The enzyme responsible for producing these molecules is a potential therapeutic target, but its i...
Article
We have recently characterized essential inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR)-mediated Ca2+ signals generated during the differentiation of slow muscle cells (SMCs) in intact zebrafish embryos. Here, we show that the lysosomal two-pore channel 2 (TPC2) also plays a crucial role in generating, and perhaps trigger...
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Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion...
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Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two pore Ca 2+ channel 2 (TPC2), a newly identified ebolavirus entry fa...
Article
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Ca2+-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca2+ responses in cardiac myocyt...
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Pancreatic beta cells are electrically excitable, and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs) which leads to the exocytosis of insulin granules. We have examined the possible role of NAADP-mediated Ca2+ release from intracellular stores during...
Article
The Ca2+-mobilizing second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), has been with us for nearly 20 years and yet we still cannot fully agree on the identity of its target Ca2+-release channel. In spite of some recent robust challenges to the idea that two-pore channels (TPCs) represent the elusive "NAADP receptor", evidence...
Article
Full-text available
Recent interest in two-pore channels (TPCs) has resulted in a variety of studies dealing with the functional role and mechanism of action of these endo-lysosomal proteins in diverse physiological processes. With the availability of mouse lines harbouring mutant alleles for Tpcn1 and/or Tpcn2 genes, several studies have made use of them to validate,...
Article
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A cytotoxic T-lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. However, these granules are more than reservoirs of secretory cytolytic proteins but may also serve as unique Ca2+ signaling hubs that autonomously generate their own sign...
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Full-text available
The second messenger NAADP triggers Ca2+ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAA...
Article
Endolysosomes are emerging as key players that generate as well as respond to intracellular Ca(2+) signals. The role of Ca(2+) in modulating acidic organelle function has long been recognized, but it is now emerging that acidic organelles also act as intracellular Ca(2+) stores; they actively sequester Ca(2+) in their lumina and release it to the c...
Conference Paper
The permeation pathway of hTPC2 (two-pore channel 2) was studied using a plasma membrane-targeted hTPC2 (hTPC2PM) and the tetravalent cation spermine. We found that intracellular spermine blocks hTPC2PM (Kd=1.5mM) and displays intrinsic voltage sensitivity under zero extracellular Na+ condition. Extracellular Na+ decreases the observed binding ener...
Article
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Postnatal skeletal muscle mass is regulated by the balance between anabolic protein synthesis and catabolic protein degradation, and muscle atrophy occurs when protein homeostasis is disrupted. Autophagy has emerged as critical in clearing dysfunctional organelles and thus in regulating protein turnover. Here we show that endolysosomal TPC2 contrib...
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Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorp...
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Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca2+ signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of th...
Article
Since the discovery of the Ca(2+) mobilizing effects of the pyridine nucleotide metabolite, nicotinic acid adenine dinucleotide phosphate (NAADP), this molecule has been demonstrated to function as a Ca(2+) mobilizing intracellular messenger in a wide range of cell types. In this review, I will briefly summarize the distinct principles behind NAADP...
Article
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Caged derivatives of Ca(2+)-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca(2+) signaling. Caged NAADP is no longer commercially available but can be synthesized in house, as described here. In brief, a stable precursor of the caging...
Article
Full-text available
Nicotinic acid adenine dinucleotide phosphate (NAADP), like the other major messengers for Ca(2+) mobilization, is passively membrane-impermeant. Instead, a cell-permeant acetoxymethyl ester derivative of NAADP (NAADP-AM) can be synthesized as described here and used to study NAADP-mediated Ca(2+) release.
Article
In addition to mobilizing Ca(2+), NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atr...
Article
Full-text available
NAADP and other Ca(2+)-mobilizing messengers are membrane impermeant and thus must be added directly to cell-free or broken-cell preparations to effect Ca(2+) release. The sea urchin egg homogenate, where the biological activity of NAADP was first reported, remains the gold standard cell-free system for studying NAADP-mediated Ca(2+) release. Here...
Article
Full-text available
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a major messenger for Ca(2+) mobilization in cells. NAADP-binding proteins are highly selective and have a strong affinity for NAADP. This is the basis of the radioreceptor binding assay, which is used to measure NAADP levels in cells and tissues and to identify cellular stimuli that use NAAD...
Article
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The genetic basis of type 2 diabetes remains incompletely defined despite the use of multiple genetic strategies. Multiparental populations such as heterogeneous stocks (HS) facilitate gene discovery by allowing fine mapping to only a few megabases, significantly decreasing the number of potential candidate genes compared to traditional mapping str...
Article
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Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, w...
Article
Full-text available
Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent endogenous Ca2+ mobilizing second messenger that is known to release Ca2+ from the acidic endolysosomal vesicles. Our previous work showed that intracellular application of NAADP led to the increase in contractions of electrically stimulated guinea pig ventricular cardiom...
Article
NAADP potently triggers Ca(2+) release from acidic lysosomal and endolysosomal Ca(2+) stores. Human two-pore channels (TPC1 and TPC2), which are located on these stores, are involved in this process, but there is controversy over whether TPC1 and TPC2 constitute the Ca(2+) release channels. We therefore examined the single-channel properties of hum...
Article
Cell homogenates provide a simple and yet powerful means of investigating the actions of Ca(2+)-mobilizing second messengers and their target Ca(2+) stores. The sea urchin egg homogenate is particularly useful and almost unique in retaining robust Ca(2+) responses to all three major messengers, i.e., inositol 1,4,5-trisphosphate (IP3), cyclic ADP-r...
Article
Much excitement surrounded the proposal that a family of endo-lysosomal channels, the two-pore channels (TPCs) were the long sought after targets of the Ca(2+) -mobilising messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, the role of TPCs in NAADP signalling may be more complex than originally envisaged. First, NAADP may no...
Article
Full-text available
Analogues of the potent Ca(2+) releasing second messenger cyclic ADP-ribose (cADPR) with a 1,2,3-triazole pyrophosphate bioisostere were synthesised by click-mediated macrocyclisation. The ability to activate Ca(2+) release was surprisingly retained, and hydrolysis of cADPR by CD38 could also be inhibited, illustrating the potential of this approac...
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Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-relat...
Article
Two-pore channels (TPC1-3) are recently identified endolysosomal ion channels. The mechanism by which these channels are regulated at the molecular level is presently unclear. To identify putative protein regulators of TPCs, we performed unbiased transcriptome-wide screens using the yeast two-hybrid technique to identify potential protein-protein i...
Article
The endoplasmic reticulum (ER) is a major organelle in all eukaryotic cells which performs multiple functions including protein and lipid synthesis and sorting, drug metabolism, and Ca(2+) storage and release. The ER, and its specialized muscle counterpart the sarcoplasmic reticulum (SR), is the largest and most extensive of Ca(2+) storage organell...
Article
Full-text available
The pyridine nucleotide second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), is widely accepted to release Ca2+ from acidic Ca2+-storing organelles (e.g., endo-lysosomes, lysosome-related organelles, exocytotic vesicles) (Guse, 2012; Morgan et al., 2011; Zhu et al., 2010) but the identity of its molecular target channel has been...
Article
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When cytotoxic T-lymphocytes (CTLs) kill infected or cancerous cells they secrete cytolytic proteins (perforin and granzymes) into the target cell. These "death factors" are pre-stored in cytolytic granules within the CTL until an increase in the intracellular Ca(2+) drives granule exocytosis. However, not all sources of Ca(2+) stimulate exocytosis...
Article
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The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca2+ stores that release Ca2+ in response to the second messengers IP3 and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca2+ released from acidic organelles by NAADP subsequently recruits IP3 or ryanodine receptors on the ER, an anterograde signal i...
Article
NAADP is a potent endogenous Ca2+-mobilising agent with actions in many cell types, including cardiac myocytes1,2. Our previous work suggests that NAADP causes Ca2+ release from an acidic endolysosomal store, leading to additional Ca2+ uptake by the SR1,2. Endolysosomal Ca2+ release is thought to occur via a two-pore Ca2+ channel (TPC), which exist...
Article
Full-text available
A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca(2+)-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the endoplasmic reticulum activates the store-operated Ca(2+)-influx pathway that is necessary...
Article
Full-text available
The regulation of Ca(2+) release by luminal Ca(2+) has been well studied for the ryanodine and IP(3) receptors but has been less clear for the NAADP-regulated channel. In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca(2+) with the membrane-permeant, low affinity Ca(2+) buffer, TPEN, and monitoring NAADP-induce...
Article
Evidence suggests that ß-Adrenergic receptor signaling increases heart rate and force through not just cyclic AMP but also the Ca(2+)-releasing second messengers NAADP (nicotinic acid adenine dinucleotide phosphate) and cADPR (cyclic ADP-ribose). Nevertheless, proof of the physiological relevance of these messengers requires direct measurements of...
Article
Two-pore channels (TPCs) constitute a family of endolysosomal cation channels with functions in Ca²⁺ signaling. We used a mutational analysis to investigate the role of channel domains for the trafficking of the Arabidopsis TPC1 to the tonoplast, a process that is generally not well understood in plants. The results show that the soluble C-terminus...
Article
Ca(2+) signals are probably the most common intracellular signaling elements, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca(2+) signals by mobilizing Ca(2+) from intracellular stores. Inositol trisphosphate (IP₃) was the first messenger shown to link event...
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Full-text available
Mammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular anal...
Article
Full-text available
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites,...
Article
Full-text available
Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion ch...
Article
Full-text available
Two-pore channels (TPCs) have been recently identified as NAADP-regulated Ca2+ release channels, which are localized on the endolysosomal system. TPCs have a 12-transmembrane domain (TMD) structure and are evolutionary intermediates between the 24-TMD α-subunits of Na+ or Ca2+ channels and the transient receptor potential channel superfamily, which...
Article
In atrial myocytes, the sarcoplasmic reticulum (SR) has an essential role in regulating the force of contraction as a consequence of its involvement in excitation-contraction coupling (ECC). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) mobilizing messenger that acts to release Ca(2+) from an acidic store in mammalian cells. The...
Article
Full-text available
Nicotinic acid dinucleotide phosphate (NAADP) is unique amongst Ca(2+) mobilizing messengers in that its principal function is to mobilize Ca(2+) from acidic organelles. Early studies indicated that it was likely that NAADP activates a novel Ca(2+) release channel distinct from the well characterized Ca(2+) release channels on the (sarco)-endoplasm...
Article
Full-text available
A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphat...
Article
We have recently demonstrated that human TPC2 is an ion-channel equipped with the specialised gating and conduction properties required to fulfil the role of the lysosomal NAADP-sensitive Ca2+-release channel (Pitt et al., 2010 J.Biol.Chem. In press). We now examine the mechanisms that underlie NAADP activation of TPC2 channels after reconstitution...