Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats

Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 microM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 microM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+,Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels.     

TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity

Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1(-/-) cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H(+) channel, and the increased lysosomal acidification in TRP-ML1(-/-) cells is likely caused by the loss of TRP-ML1-mediated H(+) leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1(-/-) cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1(-/-) cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1(-/-) cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.     

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.     

TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 downward arrow Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.     

NAADP mobilizes calcium from the endoplasmic reticular Ca(2+) store in T-lymphocytes

The target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca(2+) signaling. However, the inhibitory effect was not specific since Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H(+)-ATPase, did not block or reduce NAADP-induced Ca(2+) signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca(2+) stores, did not antagonize subsequent NAADP-induced Ca(2+) signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca(2+) signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca(2+) signaling was obtained. The sensitivity of NAADP-induced Ca(2+) signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394-21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum.     

Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV,a disorder affecting the lysosomal pathway

Mucolipin-1 (MLN1) is a membrane protein with homology to the transient receptor potential channels and other non-selective cation channels. It is encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), an autosomal recessive disease that is characterized by severe abnormalities in neurological development as well as by ophthalmologic defects. At the cellular level, MLIV is associated with abnormal lysosomal sorting and trafficking. Here we identify the channel function of human MLN1 and characterize its properties. MLN1 represents a novel Ca(2+)-permeable channel that is transiently modulated by changes in [Ca(2+)]. It is also permeable to Na(+) and K(+). Large unitary conductances were measured in the presence of these cations. With its Ca(2+) permeability and modulation by [Ca(2+)], MLN1 could play a major role in Ca(2+) transport regulating lysosomal exocytosis and potentially other phenomena related to the trafficking of late endosomes and lysosomes.     

Mucolipidosis type IV and the mucolipins

MLIV (mucolipidosis type?IV) is a neurodegenerative lysosomal storage disorder caused by mutations in MCOLN1, a gene that encodes TRPML1 (mucolipin-1), a member of the TRPML (transient receptor potential mucolipin) cation channels. Two additional homologues are TRPML2 and TRPML3 comprising the TRPML subgroup in the TRP superfamily. The three proteins play apparently key roles along the endocytosis process, and thus their cellular localization varies among the different group members. Thus TRPML1 is localized exclusively to late endosomes and lysosomes, TRPML2 is primarily located in the recycling clathrin-independent GPI (glycosylphosphatidylinositol)-anchored proteins and early endosomes, and TRPML3 is primarily located in early endosomes. Apparently, all three proteins' main physiological function underlies Ca(2+) channelling, regulating the endocytosis process. Recent findings also indicate that the three TRPML proteins form heteromeric complexes at least in some of their cellular content. The physiological role of these complexes in lysosomal function remains to be elucidated, as well as their effect on the pathophysiology of MLIV. Another open question is whether any one of the TRPMLs bears additional function in channel activity.     

Functional links between mucolipin-1 and Ca2+-dependent membrane trafficking in mucolipidosis IV

Most of the membrane trafficking phenomena including those involving the interactions between endosomes and lysosomes are regulated by changes in intracellular Ca2+ (Cai). These processes are disturbed in some types of mucolipidoses and other lysosomal storage disorders, such as mucolipidosis IV (MLIV), a neurological disorder that usually presents during the first year of life with blindness, cognitive impairment, and psychomotor delays. It is caused by mutations in MCOLN1, the gene encoding mucolipin-1 (MLN1), which we have recently established to represent a Ca2+-permeable cation channel that is transiently modulated by changes in Cai. The cells of MLIV patients contain enlarged lysosomes that are likely associated with abnormal sorting and trafficking of these and related organelles. We studied fibroblasts from MLIV patients and found disturbed Ca2+ signaling and large acidic organelles such as late endosomes and lysosomes (LEL) with altered cellular localization in these cells. The fusion between LEL vesicles in these cells was defective. This is a Ca2+-dependent process related to signaling pathways involved in regulation of Ca2+ homeostasis and trafficking. The MLN1 channels could play a key role in Ca2+ release from LEL vesicles, which triggers the fusion and trafficking of these organelles. The characterization of this MLN1-mediated Ca2+-dependent process should provide new insights into the pathophysiological mechanisms that lead to the development of MLIV and other mucolipidoses associated with similar disturbances in membrane trafficking.     

Genetic ablation of caveolin-1 modifies Ca2+ spark coupling in murine arterial smooth muscle cells

L-type, voltage-dependent calcium (Ca(2+)) channels, ryanodine-sensitive Ca(2+) release (RyR) channels, and large-conductance Ca(2+)-activated potassium (K(Ca)) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters Ca(2+) spark to K(Ca) channel coupling and Ca(2+) spark regulation by voltage-dependent Ca(2+) channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control (cav-1(+/+)) cells but were not observed in cav-1-deficient (cav-1(-/-)) cells. Ca(2+) spark and transient K(Ca) current frequency were approximately twofold higher in cav-1(-/-) than in cav-1(+/+) cells. Although voltage-dependent Ca(2+) current density was similar in cav-1(+/+) and cav-1(-/-) cells, diltiazem and Cd(2+), voltage-dependent Ca(2+) channel blockers, reduced transient K(Ca) current frequency to approximately 55% of control in cav-1(+/+) cells but did not alter transient K(Ca) current frequency in cav-1(-/-) cells. Furthermore, although K(Ca) channel density was elevated in cav-1(-/-) cells, transient K(Ca) current amplitude was similar to that in cav-1(+/+) cells. Higher Ca(2+) spark frequency in cav-1(-/-) cells was not due to elevated intracellular Ca(2+) concentration, sarcoplasmic reticulum Ca(2+) load, or nitric oxide synthase activity. Similarly, Ca(2+) spark amplitude and spread, the percentage of Ca(2+) sparks that activated a transient K(Ca) current, the amplitude relationship between sparks and transient K(Ca) currents, and K(Ca) channel conductance and apparent Ca(2+) sensitivity were similar in cav-1(+/+) and cav-1(-/-) cells. In summary, cav-1 ablation elevates Ca(2+) spark and transient K(Ca) current frequency, attenuates the coupling relationship between voltage-dependent Ca(2+) channels and RyR channels that generate Ca(2+) sparks, and elevates K(Ca) channel density but does not alter transient K(Ca) current activation by Ca(2+) sparks. These findings indicate that cav-1 is required for physiological Ca(2+) spark and transient K(Ca) current regulation in cerebral artery smooth muscle cells.     

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle

In arterial myocytes the Ca(2+) mobilizing messenger NAADP evokes spatially restricted Ca(2+) bursts from a lysosome-related store that are subsequently amplified into global Ca(2+) waves by Ca(2+)-induced Ca(2+)-release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) abolished global Ca(2+) waves but not Ca(2+) bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca(2+) signalling by NAADP.     

Membrane potential regulates nicotinic acid adenine dinucleotide phosphate (NAADP) dependence of the pH- and Ca2+-sensitive organellar two-pore channel TPC1

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger that mobilizes Ca(2+) from the acidic endolysosomes by activation of the two-pore channels TPC1 and TPC2. The channel properties of human TPC1 have not been studied before, and its cellular function is not known. In the present study, we characterized TPC1 incorporated into lipid bilayers. The native and recombinant TPC1 channels are activated by NAADP. TPC1 activity requires acidic luminal pH and high luminal Ca(2+). With Ba(2+) as the permeable ion, luminal Ca(2+) activates TPC1 with an apparent K(m) of 180 μm. TPC1 operates in two tightly coupled conductance states of 47 ± 8 and 200 ± 9 picosiemens. Importantly, opening of the large conductance markedly increases the small conductance mean open time. Changes in membrane potential from 0 to -60 mV increased linearly both the small and the large conductances and NP(o), indicating that TPC1 is regulated by voltage. Intriguingly, the apparent affinity for activation of TPC1 by its ligand NAADP is not constant. Rather, hyperpolarization increases the apparent affinity of TPC1 for NAADP by 10 nm/mV. The concerted regulation of TPC1 activity by luminal Ca(2+) and by membrane potential thus provides a potential mechanism to explain NAADP-induced Ca(2+) oscillations. These findings reveal unique properties of TPC1 to explain its role in Ca(2+) oscillations and cell function.     

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.     

Involvement of PI3K in HCV-related lymphoproliferative disorders

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.     

Critical role for CD38-mediated Ca2+ signaling in thrombin-induced procoagulant activity of mouse platelets and hemostasis

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca(2+) signal, resulting from a coordinated interplay of Ca(2+)-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca(2+) signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38(+/+) platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38(-/-) platelets. Similarly, PS exposure and Ca(2+) signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca(2+) signaling mediated by its products, cADPR and NAADP.     

Lysosome-sarcoplasmic reticulum junctions. A trigger zone for calcium signaling by nicotinic acid adenine dinucleotide phosphate and endothelin-1

Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca(2+) signals by mobilizing thapsigargin-insensitive stores. Such localized Ca(2+) signals may initiate global Ca(2+) waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca(2+)-induced Ca(2+) release. Here we show that NAADP evokes localized Ca(2+) signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca(2+) store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADP-dependent Ca(2+) signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca(2+) stores may underpin the generation of differential Ca(2+) signaling patterns by different Ca(2+)-mobilizing messengers.     

Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca(2+) in several types of cells. We have used Ca(2+)-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca(2+) in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca(2+) rise in up to 90% of astrocytes in culture (EC(50)=7 microM). The NAADP-evoked Ca(2+) rise was maintained in the absence of extracellular Ca(2+), but was suppressed after depleting the Ca(2+) stores of the ER with ATP (20 microM), with cyclopiazonic acid (10 microM) or with ionomycin (5 microM). P(2) receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid (PPADS, 100 microM), IP(3) receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 microM) and PLC inhibitor U73122 (10 microM) also reduced or suppressed the NAADP-evoked Ca(2+) rise. NAADP still evoked a Ca(2+) response after application of glycyl-l-phenylalanine-beta-naphthylamide (GPN, 200 microM), which permeabilizes lysosomes, or preincubation with H(+)-ATPase inhibitor bafilomycin A1 (4 microM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 microM), that impairs mitochondrial Ca(2+) handling. In acute brain slices, NAADP (10 microM) evoked Ca(2+) transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in mammalian astrocytes, at least partly by activating metabotropic P(2)Y receptors.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease

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 channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling.     

Identification of the Penta-EF-hand Protein ALG-2 as a Ca2+-dependent Interactor of Mucolipin-1

Loss of function mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmological abnormalities. MCOLN1 is an ion channel that regulates membrane transport along the endolysosomal pathway. It has been suggested that MCOLN1 participates in several Ca²⁺-dependent processes, including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Here, we searched for proteins that interact with MCOLN1 in a Ca²⁺-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca²⁺, and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37 and 49. We further show that MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by overexpression of an ATPase-defective dominant-negative form of Vps4B (Vps4B^E235Q). In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that overexpression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosome markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. Overall, our data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca²⁺ sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway.     

Toxin pores endocytosed during plasma membrane repair traffic into the lumen of MVBs for degradation

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.