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1.
Cell Calcium ; 116: 102801, 2023 12.
Article in English | MEDLINE | ID: mdl-37742482

ABSTRACT

Multiple families of Ca2+-permeable channels co-exist on lysosomal Ca2+ stores but how each family couples to its own unique downstream physiology is unclear. We have therefore investigated the Ca2+-signalling architecture underpinning different channels on the same vesicle that drive separate pathways, using phagocytosis as a physiological stimulus. Lysosomal Ca2+-channels are a major Ca2+ source driving particle uptake in macrophages, but different channels drive different aspects of Fc-receptor-mediated phagocytosis: TPC2 couples to dynamin activation, whilst TRPML1 couples to lysosomal exocytosis. We hypothesised that they are driven by discrete local plumes of Ca2+ around open channels (Ca2+ nanodomains). To test this, we optimized Ca2+-nanodomain recordings by screening panels of genetically encoded Ca2+ indicators (GECIs) fused to TPC2 to monitor the [Ca2+] next to the channel. Signal calibration accounting for the distance of the GECI from the channel mouth reveals that, during phagocytosis, TPC2 generates local Ca2+ nanodomains around itself of up to 42 µM, nearly a hundred-fold greater than the global cytosolic [Ca2+] rise. We further show that TPC2 and TRPML1, though on the same lysosomes, generate autonomous Ca2+ nanodomains of high [Ca2+] that are largely insulated from one another, a platform allowing their discrete Ca2+-decoding to promote unique respective physiologies.


Subject(s)
Transient Receptor Potential Channels , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolism , Lysosomes/metabolism , Phagocytosis , Signal Transduction , Exocytosis
2.
Handb Exp Pharmacol ; 278: 3-34, 2023.
Article in English | MEDLINE | ID: mdl-35879580

ABSTRACT

The discovery of NAADP-evoked Ca2+ release in sea urchin eggs and then as a ubiquitous Ca2+ mobilizing messenger has introduced several novel paradigms to our understanding of Ca2+ signalling, not least in providing a link between cell stimulation and Ca2+ release from lysosomes and other acidic Ca2+ storage organelles. In addition, the hallmark concentration-response relationship of NAADP-mediated Ca2+ release, shaped by striking activation/desensitization mechanisms, influences its actions as an intracellular messenger. There has been recent progress in our understanding of the molecular mechanisms underlying NAADP-evoked Ca2+ release, such as the identification of the endo-lysosomal two-pore channel family of cation channels (TPCs) as their principal target and the identity of NAADP-binding proteins that complex with them. The NAADP/TPC signalling axis has gained recent prominence in pathophysiology for their roles in such disease processes as neurodegeneration, tumorigenesis and cellular viral entry.


Subject(s)
Calcium Channels , Calcium , Humans , Calcium Channels/metabolism , Calcium/metabolism , Signal Transduction , NADP/metabolism , Lysosomes/metabolism , Calcium Signaling/physiology
3.
Biochem Soc Trans ; 50(4): 1143-1155, 2022 08 31.
Article in English | MEDLINE | ID: mdl-35959977

ABSTRACT

In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PI(3,5)P2, TPCs are an integral part of fundamental signal-transduction pathways, but their array and plasticity of cation conductances (Na+, Ca2+, H+) allow them to variously signal electrically, osmotically or chemically. Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests.


Subject(s)
Calcium Channels , Calcium , Calcium/metabolism , Calcium Channels/metabolism , Calcium Signaling , Lysosomes/metabolism , NADP/metabolism , Signal Transduction
4.
Cell Calcium ; 101: 102516, 2022 01.
Article in English | MEDLINE | ID: mdl-34922066

ABSTRACT

Acidic organelles act as intracellular Ca2+ stores; they actively sequester Ca2+ in their lumina and release it to the cytosol upon activation of endo-lysosomal Ca2+ channels. Recent data suggest important roles of endo-lysosomal Ca2+ channels, the Two-Pore Channels (TPCs) and the TRPML channels (mucolipins), in different aspects of immune-cell function, particularly impacting membrane trafficking, vesicle fusion/fission and secretion. Remarkably, different channels on the same acidic vesicles can couple to different downstream physiology. Endo-lysosomal Ca2+ stores can act under different modalities, be they acting alone (via local Ca2+ nanodomains around TPCs/TRPMLs) or in conjunction with the ER Ca2+ store (to either promote or suppress global ER Ca2+ release). These different modalities impinge upon functions as broad as phagocytosis, cell-killing, anaphylaxis, immune memory, thrombostasis, and chemotaxis.


Subject(s)
Endosomes , Lysosomes , Biological Transport , Calcium/metabolism , Calcium Signaling , Endosomes/metabolism , Lysosomes/metabolism , NADP/metabolism , Phagocytosis
5.
Biochim Biophys Acta Mol Cell Res ; 1868(7): 119040, 2021 06.
Article in English | MEDLINE | ID: mdl-33872669

ABSTRACT

The emergence of endo-lysosomes as ubiquitous Ca2+ stores with their unique cohort of channels has resulted in their being implicated in a growing number of processes in an ever-increasing number of cell types. The architectural and regulatory constraints of these acidic Ca2+ stores distinguishes them from other larger Ca2+ sources such as the ER and influx across the plasma membrane. In view of recent advances in the understanding of the modes of operation, we discuss phagocytosis as a template for how endo-lysosomal Ca2+ signals (generated via TPC and TRPML channels) can be integrated in multiple sophisticated ways into biological processes. Phagocytosis illustrates how different endo-lysosomal Ca2+ signals drive different phases of a process, and how these can be altered by disease or infection.


Subject(s)
Calcium/metabolism , Lysosomes/metabolism , Phagosomes/metabolism , Animals , Calcium Channels/metabolism , Calcium Signaling/physiology , Cell Membrane/metabolism , Endosomes/metabolism , Humans , NADP/metabolism , Phagocytosis/physiology , Phagosomes/physiology , Transient Receptor Potential Channels/metabolism
6.
J Cell Sci ; 134(2)2021 01 27.
Article in English | MEDLINE | ID: mdl-33328326

ABSTRACT

Pharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca2+ entry (SOCE) by these agents. Dipeptides [glycyl-L-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester] that are inducers of lysosomal membrane permeabilization (LMP) uncoupled endoplasmic reticulum Ca2+-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai. Similarly, the K+/H+ ionophore, nigericin, that rapidly elevates lysosomal pH, also inhibited SOCE in a Stim1-dependent manner. In contrast, other strategies for manipulating lysosomes (bafilomycin A1, lysosomal re-positioning) had no effect upon SOCE. Finally, the effects of GPN on SOCE and Stim1 was reversed by a dynamin inhibitor, dynasore. Our data show that lysosomal agents not only release Ca2+ from stores but also uncouple this release from the normal recruitment of Ca2+ influx.


Subject(s)
Calcium , Membrane Proteins , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Lysosomes/metabolism , Membrane Proteins/metabolism , ORAI1 Protein , Stromal Interaction Molecule 1
7.
Cell Calcium ; 93: 102328, 2021 01.
Article in English | MEDLINE | ID: mdl-33352478

ABSTRACT

LC3-lipidation is activated by lysosomal damage by mechanisms that are unknown and divergent from canonical autophagy. In this study, Nakamura et al, show that lysosomal damage induced by lysosomotropic agents or oxalate in renal proximal tubule cells causes lipidated LC3 to insert into the lysosomal membrane to activate TRPML1 channels and release Ca2+ from lysosomes. This leads to TFEB dephosphorylation and translocation into the nucleus which results in clearance of damaged lysosomes and their contents which may reduce the deleterious effects of crystal nephropathy.


Subject(s)
Transient Receptor Potential Channels , Autophagy , Calcium/metabolism , Kidney/metabolism , Lysosomes/metabolism
8.
EMBO J ; 39(14): e104058, 2020 07 15.
Article in English | MEDLINE | ID: mdl-32510172

ABSTRACT

Macrophages clear pathogens by phagocytosis and lysosomes that fuse with phagosomes are traditionally regarded as to a source of membranes and luminal degradative enzymes. Here, we reveal that endo-lysosomes act as platforms for a new phagocytic signalling pathway in which FcγR activation recruits the second messenger NAADP and thereby promotes the opening of Ca2+ -permeable two-pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo-lysosomal Ca2+ nanodomains rather than global cytoplasmic or ER Ca2+ signals. Motile endolysosomes contact nascent phagosomes to promote phagocytosis, whereas endo-lysosome immobilization prevents it. We show that TPC-released Ca2+ rapidly activates calcineurin, which in turn dephosphorylates and activates the GTPase dynamin-2. Finally, we find that different endo-lysosomal Ca2+ channels play diverse roles, with TPCs providing a universal phagocytic signal for a wide range of particles and TRPML1 being only required for phagocytosis of large targets.


Subject(s)
Calcineurin/metabolism , Dynamin II/metabolism , Endosomes/metabolism , Lysosomes/metabolism , Macrophages/metabolism , NADP/analogs & derivatives , Phagocytosis , Animals , Calcineurin/genetics , Dynamin II/genetics , Endosomes/genetics , Lysosomes/genetics , Mice , Mice, Knockout , NADP/metabolism
9.
Cell Calcium ; 86: 102139, 2020 03.
Article in English | MEDLINE | ID: mdl-31881482

ABSTRACT

Lysosomotropic agents have been used to permeabilize lysosomes and thereby implicate these organelles in diverse cellular processes. Since lysosomes are Ca2+ stores, this rupturing action, particularly that induced by GPN, has also been used to rapidly release Ca2+ from lysosomes. However, a recent study has questioned the mechanism of action of GPN and concluded that, acutely, it does not permeabilize lysosomes but releases Ca2+ directly from the ER instead. We therefore appraise these provocative findings in the context of the existing literature. We suggest that further work is required to unequivocally rule out lysosomes as contributors to GPN-evoked Ca2+ signals.


Subject(s)
Calcium/metabolism , Lysosomes/metabolism , Animals , Calcium Signaling/drug effects , Dipeptides/chemistry , Dipeptides/pharmacology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Humans , Lysosomes/drug effects , Models, Biological
10.
Wellcome Open Res ; 1: 18, 2016 Nov 18.
Article in English | MEDLINE | ID: mdl-28008422

ABSTRACT

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 understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. METHODS: The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. RESULTS: Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. CONCLUSION: These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

11.
Biochem Soc Trans ; 44(2): 546-53, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-27068968

ABSTRACT

Extracellular stimuli evoke the synthesis of intracellular second messengers, several of which couple to the release of Ca(2+)from Ca(2+)-storing organelles via activation of cognate organellar Ca(2+)-channel complexes. The archetype is the inositol 1,4,5-trisphosphate (IP3) and IP3receptor (IP3R) on the endoplasmic reticulum (ER). A less understood, parallel Ca(2+)signalling cascade is that involving the messenger nicotinic acid adenine dinucleotide phosphate (NAADP) that couples to Ca(2+)release from acidic Ca(2+)stores [e.g. endo-lysosomes, secretory vesicles, lysosome-related organelles (LROs)]. NAADP-induced Ca(2+)release absolutely requires organellar TPCs (two-pore channels). This review discusses how ER and acidic Ca(2+)stores physically and functionally interact to generate and shape global and local Ca(2+)signals, with particular emphasis on the two-way dialogue between these two organelles.


Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Acids , Animals , Calcium Signaling , Hydrogen-Ion Concentration , Organelles/metabolism
12.
Messenger (Los Angel) ; 5(1-2): 92-99, 2016 Jun 01.
Article in English | MEDLINE | ID: mdl-28758053

ABSTRACT

Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), in cardiac cells can trigger ventricular arrhythmias especially in failing hearts. SOICR occurs when RyRs are activated by an increase in sarcoplasmic reticulum (SR) luminal Ca2+. Carvedilol is one of the most effective drugs for preventing arrhythmias in patients with heart failure. Furthermore, carvedilol analogues with minimal ß-blocking activity also block SOICR showing that SOICR-inhibiting activity is distinct from that for ß-block. We show here that carvedilol is a potent inhibitor of cADPR-induced Ca2+ release in sea urchin egg homogenate. In addition, the carvedilol analog VK-II-86 with minimal ß-blocking activity also suppresses cADPR-induced Ca2+ release. Carvedilol appeared to be a non-competitive antagonist of cADPR and could also suppress Ca2+ release by caffeine. These results are consistent with cADPR releasing Ca2+ in sea urchin eggs by sensitizing RyRs to Ca2+ involving a luminal Ca2+ activation mechanism. In addition to action on the RyR, we also observed inhibition of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release by carvedilol suggesting a common mechanism between these evolutionarily related and conserved Ca2+ release channels.

13.
J Biol Chem ; 290(35): 21376-92, 2015 Aug 28.
Article in English | MEDLINE | ID: mdl-26152717

ABSTRACT

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.


Subject(s)
Calcium Channels/metabolism , Endosomes/metabolism , Insulin-Secreting Cells/metabolism , NADP/analogs & derivatives , Animals , Calcium/metabolism , Calcium Channels/genetics , Cells, Cultured , Glucose/metabolism , Insulin/metabolism , Insulin-Secreting Cells/cytology , Male , Membrane Potentials , Mice , Mice, Knockout , NADP/metabolism
14.
Biochem Soc Trans ; 43(3): 384-9, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26009180

ABSTRACT

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 continues to accumulate that TPCs are important for NAADP-mediated responses. This article will briefly outline the background and review more recent work pertaining to the TPC story.


Subject(s)
Calcium Channels/genetics , Calcium Signaling/genetics , Calcium/metabolism , NADP/analogs & derivatives , Animals , Calcium Channels/chemistry , Calcium Channels/metabolism , Endosomes/metabolism , Lipid Metabolism/genetics , Lysosomes/metabolism , Membrane Potentials/genetics , NADP/chemistry , NADP/genetics , NADP/metabolism
15.
EMBO J ; 34(13): 1743-58, 2015 Jul 02.
Article in English | MEDLINE | ID: mdl-25872774

ABSTRACT

The second messenger NAADP triggers Ca(2+) 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 NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.


Subject(s)
Calcium Channels/genetics , Calcium/metabolism , NADP/analogs & derivatives , Animals , Calcium Channels/metabolism , Calcium Signaling/drug effects , Calcium Signaling/genetics , Cells, Cultured , Evoked Potentials/drug effects , Gene Expression/physiology , Hydrogen-Ion Concentration , Lysosomes/drug effects , Lysosomes/physiology , Mice , Mice, Knockout , NADP/metabolism , NADP/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Signal Transduction/drug effects
16.
Methods Cell Biol ; 126: 159-95, 2015.
Article in English | MEDLINE | ID: mdl-25665446

ABSTRACT

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 cytosol upon activation of endolysosomal Ca(2+) channels. This local Ca(2+) signal is crucial for endolysosomal function and/or global Ca(2+) signaling. Importantly, defects in endolysosomal Ca(2+) are associated with disease. This chapter discusses several complimentary approaches to monitor endolysosomal Ca(2+), with particular emphasis on the inherent pitfalls that can plague the unwary.


Subject(s)
Calcium/metabolism , Lysosomes/metabolism , Animals , Calcium Signaling , Cells, Cultured , Endosomes/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Homeostasis , Humans , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Staining and Labeling
17.
Cold Spring Harb Protoc ; 2014(10): pdb.prot076927, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25275101

ABSTRACT

Nicotinic acid adenine dinucleotide phosphate (NAADP), like the other major messengers for Ca²âº 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 Ca²âº release.


Subject(s)
NADP/analogs & derivatives , Animals , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cells, Cultured , Humans , NADP/biosynthesis , NADP/chemistry , NADP/metabolism , NADP/pharmacokinetics , Permeability/drug effects
18.
Cold Spring Harb Protoc ; 2014(10): pdb.prot076935, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25275102

ABSTRACT

In addition to mobilizing Ca²âº, 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 atrial myocytes and pancreatic acinar cells). Although the connection between Ca²âº mobilization and increase in pHL is not understood, pHL can be a useful parameter to measure when studying NAADP-mediated signaling. This protocol describes the fluorescent measurement of pHL of acidic stores. It relies on the use of acridine orange (AO), a standard dye for pHL. AO selectively accumulates to high concentrations in the lumen of organelles as a function of acidity; at these high concentrations it self-quenches. When pHL increases, some AO is lost from the vesicle. As a result, the lower luminal AO concentration relieves the quenching and fluorescence increases in the lumen.


Subject(s)
Acids/metabolism , Hydrogen-Ion Concentration , NADP/analogs & derivatives , Organelles/metabolism , Weights and Measures , Animals , Male , NADP/metabolism , Sea Urchins , Signal Transduction , Spermatozoa/metabolism , Spermatozoa/ultrastructure
19.
Cold Spring Harb Protoc ; 2014(10): pdb.prot076943, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25275103

ABSTRACT

Caged derivatives of Ca²âº-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca²âº 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 reagent is made and converted to an unstable reactive reagent immediately before addition to the compound to be caged.


Subject(s)
Calcium/metabolism , NADP/analogs & derivatives , Photochemistry , Photolysis , Animals , Calcium Signaling , NADP/chemical synthesis , NADP/metabolism
20.
Cold Spring Harb Protoc ; 2014(9): 988-92, 2014 Sep 02.
Article in English | MEDLINE | ID: mdl-25183812

ABSTRACT

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 we describe how to prepare sea urchin egg homogenate and use it to measure NAADP-mediated Ca(2+) release.


Subject(s)
Calcium/metabolism , Cell Fractionation/methods , NADP/analogs & derivatives , Ovum/drug effects , Ovum/metabolism , Adenosine Triphosphate/pharmacology , Aniline Compounds , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Female , Inositol 1,4,5-Trisphosphate/pharmacology , Male , NADP/metabolism , NADP/pharmacology , Ovum/ultrastructure , Sea Urchins , Xanthenes
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