Until recently, the mechanisms that regulate endolysosomal calcium homoeostasis were poorly understood. The discovery of the molecular target of NAADP (nicotinic acid–adenine dinucleotide phosphate) as the two-pore channels resident in the endolysosomal system has highlighted this compartment as an important calcium store. The recent findings that dysfunctional NAADP release leads to defective endocytic function which in turn results in secondary lipid accumulation in the lysosomal storage disease Niemann–Pick type C, is the first evidence of a direct connection between a human disease and defective lysosomal calcium release. In the present review, we provide a summary of the current knowledge on mechanisms of calcium homoeostasis within the endolysosomal system and how these mechanisms may be affected in human metabolic disorders.

Importance of calcium

Cellular calcium signalling is tightly regulated, with a number of intracellular stores that maintain high concentrations of calcium, such as the ER (endoplasmic reticulum), which has an estimated calcium content of ~0.5–2 mM [1,2], compared with the cytosol, which is maintained at a constant low calcium concentration (~100 nM [3]), although this can change rapidly in response to numerous intra- and extra-cellular signals and processes [4]. Numerous protein channels, co-factors and pumps are responsible for regulating and maintaining these concentrations, with other channels functioning to release calcium in response to ligands such as the intracellular second messengers NAADP (nicotinic acid–adenine dinucleotide phosphate), InsP3 and cADPR (cADP-ribose) [4]. One cellular compartment that has recently emerged as a novel calcium store that is important in cellular calcium signalling is the lysosome [4]. In the present review, we summarize current knowledge on the normal regulation of this store, the requirement for lysosomal calcium in initiating local and global calcium changes for normal cellular function and the disease phenotypes that arise when these processes are aberrant.

Endolysosomal calcium store filling

To date, two accurate estimates of lysosomal calcium concentration in the acidic compartment have been made utilizing low-affinity calcium probes conjugated to high-molecular-mass dextran [5,6]. In order to accurately estimate the intralysosomal calcium concentrations, the Kd of the calcium probe requires adjusting to the exact lysosomal pH. Both studies (utilizing different calcium probes) estimated the calcium content of macrophage lysosomes to be ~400–600 μM [5,6]. Human fibroblast lysosomes were estimated to have ~550 μM calcium [6]. The findings of these studies, which are in agreement with earlier indirect measurements [7], indicate that the lysosome is a substantial calcium store not dissimilar in concentration to that of the ER [2]. The majority of calcium in the lysosome is not delivered there by endocytosis, as adjusting the extracellular calcium concentration from 2 mM to 0.5 mM had no effect on the lysosomal calcium concentration [5]. Further lowering to 0.1 mM marginally reduced the lysosomal calcium concentration, indicating that endocytosis does provide a small quantity of calcium to the lysosome [5], a source that has been shown to be essential for vacuole fusion in yeast [8]. This is in agreement with another study [9] showing that the majority of endocytosed calcium is released into the cytosol from early endosomes in response to the initiation of acidification of the endolysosomal system (Figure 1). This also appears to be the case during pinocytosis where a decrease in pH from 7.2 to 6.2 is coupled with a fall of two orders of magnitude in calcium concentration, illustrating further that the early-endosome calcium concentration is low [5]. These studies indicate that the extracellular calcium concentration (~1 mM) does not reflect the early-endosomal calcium concentration, which has been estimated in two studies to be in the range 3–40 μM [9,10].

Endolysosomal calcium concentrations and pH

Figure 1
Endolysosomal calcium concentrations and pH

Calcium can be transported from the extracellular fluid into early endosomes by endocytosis. Early endosomes are depleted of calcium in response to acidification. Late endosomes and lysosomes are filled with calcium via a proton-dependent mechanism. Local elevations in calcium are required for fusion and transport between endocytic compartments.

Figure 1
Endolysosomal calcium concentrations and pH

Calcium can be transported from the extracellular fluid into early endosomes by endocytosis. Early endosomes are depleted of calcium in response to acidification. Late endosomes and lysosomes are filled with calcium via a proton-dependent mechanism. Local elevations in calcium are required for fusion and transport between endocytic compartments.

Entry of calcium into the lysosome is a proton-dependent process [5], as deprotonation of the lysosome by inhibitors of the vacuolar ATPase (bafilomycin) or proton ionophores (ammonium) results in loss of lysosomal calcium proportional to the extent/speed of deacidification [5]. This loss of calcium, in turn, prevents the reformation of lysosomes from hybrid late-endosome–lysosome compartments [11]. The proteins that regulate calcium entry into lysosomes in mammalian cells remain unknown. Model organisms such as yeast and Arabidopsis, however, are known to have H+/Ca2+ exchangers (not present in mammals) and calcium-transporting ATPases in the vacuole (the activity of which have been characterized in mammalian cells) [12,13]. One such Ca2+-ATPase that has been suggested to fill platelet dense-core granules (lysosome-related organelles) is SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3a [14] (Figure 2). Three studies have shown that inhibiting SERCA3a activity with TBHQ (t-butylhydroquinone) depletes NAADP-mediated calcium release from these compartments and that calcium entry via SERCA3a into platelet dense-core granules is dependent on store-operated calcium entry, via Stim1 (stromal interaction molecule 1) [1416]. Whether SERCA3a is responsible for lysosomal calcium entry or is specific to platelet dense-core granules is unknown, but it is tempting to speculate that this channel may emerge as the Ca2+-ATPase activity reported in purified lysosomal preparations [12], which is highly sensitive to changes in luminal concentrations of other bivalent cations, including lead [17]. A comprehensive study in sea urchin egg homogenate identified that the sea urchin reserve granule (equivalent to lysosomes) calcium-entry proteins require ATP and a proton gradient, but are insensitive to thapsigargin and only slightly sensitive to vanadate [18]. These findings suggest that P-type ATPases do not play a major role in lysosomal calcium entry [18]. Instead, calcium exchangers, including Ca2+/H+ or, more likely (owing to the absence of calcium exchangers in mammals [13]), Na+/H+ coupled to Na+/Ca2+ exchangers, are likely to be important [18] (Figure 2). These have already been observed in melanosomes (lysosome-related organelles), and when dysfunctional lead to defects in melanosome biogenesis [19]. Although the exact identity of these channels remains a mystery, proteomics studies on purified lysosomal membranes have identified potential candidates [20].

Lysosomal calcium channels

Figure 2
Lysosomal calcium channels

Acidic stores are filled with calcium via the action of as yet unidentified ion transporting ATPases or solute channels (SLCs). SLCs may transport calcium directly against the proton gradient or this may be coupled to sodium transport (Na+/H+ and Na+/Ca2+ transporter). Calcium release is mediated by NAADP-sensitive TPCs and, potentially, TRPML1. Defects in TRPML1 that result in the human LSD MLIV lead to enlarged endolysosomes and defective recycling of lipids, suggesting a potential defect in endosomal fission. Defects in the NPC1 protein lead to the accumulation of sphingosine (sph) that inhibits calcium entry into lysosomes. This in turn depletes the acidic calcium store and subsequently leads to defective calcium release from TPCs, which results in defective endolysosome fusion and storage of lipids.

Figure 2
Lysosomal calcium channels

Acidic stores are filled with calcium via the action of as yet unidentified ion transporting ATPases or solute channels (SLCs). SLCs may transport calcium directly against the proton gradient or this may be coupled to sodium transport (Na+/H+ and Na+/Ca2+ transporter). Calcium release is mediated by NAADP-sensitive TPCs and, potentially, TRPML1. Defects in TRPML1 that result in the human LSD MLIV lead to enlarged endolysosomes and defective recycling of lipids, suggesting a potential defect in endosomal fission. Defects in the NPC1 protein lead to the accumulation of sphingosine (sph) that inhibits calcium entry into lysosomes. This in turn depletes the acidic calcium store and subsequently leads to defective calcium release from TPCs, which results in defective endolysosome fusion and storage of lipids.

Endolysosomal calcium store release

Calcium release from lysosomes is an essential cellular process (Table 1) regulating endolysosome function [11,21]. It was reported recently that the calcium-release channel for the second messenger NAADP is the TPC (two-pore channel) family of proteins [22] (Figure 2) that reside in the endolysosomal system [21]. The discovery that NAADP acts on a lysosomal receptor highlights further the emergence of the lysosome as a bona fide calcium-signalling store. NAADP is the most potent calcium-signalling second messenger [4]. Intracellular calcium signalling mediated by NAADP is important in multiple cellular functions, including triggering of the acrosome reaction during fertilization, endocytosis, insulin secretion and neuronal growth [22a]. Lysosomal calcium release, coupled to a transient increase in lysosomal pH, triggered by NAADP, can be potentiated by subsequent ER calcium release [23,24]. The discovery of TPCs as the receptors for NAADP suggests that the other candidate channel, TRPML (transient receptor potential mucolipin) 1 [25], is not an NAADP receptor, as we and others have reported [26,27] (Figure 2). We have recently found that lysosomal calcium release in response to NAADP-AM (NAADP acetoxymethyl ester) from TRPML1-null cells is not reduced, indicating that this receptor is not involved in NAADP-mediated calcium signaling (K. Peterneva, F.M. Platt and E. Lloyd-Evans unpublished work). Furthermore, the high concentrations of NAADP required to release calcium from another candidate NAADP receptor, TRPM2 (transient receptor potential melastatin 2) [28], would again suggest that this channel is not primarily involved in NAADP-mediated calcium release from lysosomes. However, it would appear that the TRPML channels are indeed regulators of lysosomal calcium release [29,30], although they are permeable to multiple univalent ions, including Na+, K+ and, potentially, H+ [31]. An endogenous stimulus to initiate ion flux has yet to be discovered, although some chemical tools have recently emerged [32]. TRPML channels, in a similar manner to TPCs, may have complex roles in regulating endosome function that is described further below. Other channels involved in ER calcium release have been implicated in calcium release from lysosomes [7], although these channels have not been reported in proteomic studies of lysosomal membranes [20]. This discrepancy could potentially arise from contamination of subcellular fractions by other organelles, or even autophagy-driven transport of other membranes to the lysosome.

Table 1
Functional importance of lysosomal calcium release
Function Significance Reference(s) 
Endolysosome transport Sorting and recycling of endocytosed material [6,21,27,30
Endolysosome fusion Delivery of material, degradation [6,8,11
Phagolysosome fusion/autolysosome fusion Clearance of infectious agents, clearance of defective organelles [35,37,39
Lysosome-related organelle biogenesis Pigmentation (melanosome), T-cell function, platelet dense-body formation [19,51
Lysosomal exocytosis Wound healing [52
Phosphatidylserine externalization Initiating factor in apoptosis [57
Function Significance Reference(s) 
Endolysosome transport Sorting and recycling of endocytosed material [6,21,27,30
Endolysosome fusion Delivery of material, degradation [6,8,11
Phagolysosome fusion/autolysosome fusion Clearance of infectious agents, clearance of defective organelles [35,37,39
Lysosome-related organelle biogenesis Pigmentation (melanosome), T-cell function, platelet dense-body formation [19,51
Lysosomal exocytosis Wound healing [52
Phosphatidylserine externalization Initiating factor in apoptosis [57

Importance of endolysosomal calcium in regulating the function of the endocytic system

Calcium release is required to trigger the final step in fusion between docked endosomes and lysosomes (between 10 and 100 nm apart) post-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex assembly [6,33]. Local calcium release from luminal stores is essential as, in a cell-free system, fast calcium quenching with BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid] inhibits late-endosome–lysosome fusion, whereas slow EGTA-induced calcium quenching does not [11], and fusion is modulated at least in part by the action of calcium-bound calmodulin [8]. It is estimated that between 0.1 and 1 μM calcium is required for fusion to occur and that high concentrations of extralysosomal calcium (0.1–1 mM) are inhibitory [11]. Chelating intravesicular calcium with EGTA-AM or high-affinity rhodamine–dextran inhibits vesicular fusion and leads to a disease phenotype similar to NPC (Niemann–Pick type C) disease described below [5,6]. Interestingly, overexpression of the NAADP receptors TPC1 or TPC2 leads to enhanced lysosomal calcium release and subsequent defects in endocytosis similar to those seen in LSDs (lysosomal storage diseases) [21], suggesting that normal endocytic function is coupled to tightly regulated lysosomal calcium homoeostasis.

Whereas intraluminal calcium release is necessary for endolysosome fusion, very little is known about the mechanisms that lead to calcium-dependent phagolysosome and autophagic vacuole–lysosome (autolysosome) fusion. Defects in intracellular calcium signalling are known to contribute to defective clearance of Mycobacterium tuberculosis [34] and Listeria monocytogenes [35], whereas chelation of intracellular calcium leads to inhibition of cellular autophagy [36]. Considering the requirement for local calcium release in order to fuse endosomes and lysosomes, it is interesting to speculate whether lysosomal calcium release might regulate phagolysosome and autolysosome fusion, as both processes are known to involve alterations in intracellular calcium [36,37]. The lysosomal calcium defect observed in NPC cells may lead to the reported defects in phagolysosome clearance [38], whereas lysosomal calcium signalling may also play a role in defective autophagy seen in MLIV (mucolipidosis IV) [39]. Accurate estimation of phagosomal and autophagic vacuole calcium concentration would aid in our understanding of these processes.

Diseases with altered endolysosomal calcium homoeostasis

To date, very few diseases have been associated directly with defects in lysosomal calcium. Those that have are predominantly LSDs. Whereas some of these disorders have been associated with defective intracellular calcium homoeostasis associated with the ER (Table 2) [40,41], the subset of LSDs that are caused by mutations in lysosomal transmembrane proteins appear to have predominantly lysosomal calcium homoeostasis defects (Table 2). These are summarized below.

Table 2
Lysosomal disorders with defective calcium homoeostasis
Disease Storage material Calcium defect Reference 
Gaucher Glucosylceramide Enhanced ryanodine receptor activity (ER) [40
Sandhoff GM2 ganglioside Decreased SERCA activity (ER) [41
GM1-gangliosidosis GM1 ganglioside Decreased SERCA activity (ER) [41
Niemann–Pick type A Sphingomyelin Altered ER channel levels [58
NPC Sphingosine, sphingomyelin, cholesterol, glycosphingolipids Reduced lysosomal calcium [6
MLIV Mucopolysaccharides, glycosphingolipids, phospholipids, lipofuscin Unknown [31
CHS Unknown Enhanced lysosomal calcium entry [53
Mucopolysaccharidosis type 1 Glycosaminoglycans Increased lysosomal calcium [54
Disease Storage material Calcium defect Reference 
Gaucher Glucosylceramide Enhanced ryanodine receptor activity (ER) [40
Sandhoff GM2 ganglioside Decreased SERCA activity (ER) [41
GM1-gangliosidosis GM1 ganglioside Decreased SERCA activity (ER) [41
Niemann–Pick type A Sphingomyelin Altered ER channel levels [58
NPC Sphingosine, sphingomyelin, cholesterol, glycosphingolipids Reduced lysosomal calcium [6
MLIV Mucopolysaccharides, glycosphingolipids, phospholipids, lipofuscin Unknown [31
CHS Unknown Enhanced lysosomal calcium entry [53
Mucopolysaccharidosis type 1 Glycosaminoglycans Increased lysosomal calcium [54

Niemann–Pick type C1 disease

NPC disease is an autosomal recessive neurodegenerative LSD [42]. NPC is caused by mutation in one of two genes, NPC1 (95% of cases) and NPC2 (5% of cases), leading to identical clinical phenotypes [42]. NPC cells are characterized by lysosomal storage of multiple lipids, including sphingosine, sphingomyelin, cholesterol, glycosphingolipids and lyso(bis)phosphatidic acid [42]. NPC disease is classically regarded as a lysosomal cholesterol storage disease [42]. We recently reported that the first event in NPC disease pathogenesis is the lysosomal accumulation of sphingosine, a product of lysosomal catabolism of ceramide [6]. Upon generation, sphingosine is protonated and requires a transporter for lysosomal egress [42], which we hypothesize to be the NPC1 protein, also associated with transport of other amines [43]. Lysosomal storage of sphingosine in NPC cells leads to a defect in filling of the acidic store with calcium that, in turn, disrupts NAADP-mediated calcium release (NPC is the first disease to be associated with abnormal NAADP calcium signalling) [6]. This abnormal acidic store calcium homoeostasis is responsible for the defects in late-endosome–lysosome fusion reported in NPC cells and results in secondary storage of lipids, including cholesterol, sphingomyelin and glycosphingolipids [6]. Interestingly, this defect in acidic store calcium filling occurs independently of pH, as NPC lysosomes have no acidification defect [6]. This is in agreement with the results of a previous study that demonstrates no alteration in pH following reduction in acidic store calcium with low calcium buffers [5]. Enhancing cytosolic calcium with curcumin (weak SERCA antagonist) corrects multiple cellular NPC defects, including cholesterol storage, further illustrating that this defect is secondary to the primary sphingosine accumulation (the levels of which are unaltered by curcumin) [6]. The beneficial effects of curcumin can be inhibited by the membrane permeant calcium chelator BAPTA-AM, indicating that curcumin mediates its beneficial effect on NPC cells and tissues in a calcium-signalling-dependent manner [6]. As a result of the increased calcium concentration around the lysosome, fusion between late endosomes and lysosomes in NPC cells occurs rapidly, and lipids can exit the system, correcting the disease phenotypes in vitro and in vivo in a mouse model of NPC disease [6]. Interestingly, the elevation in cytosolic calcium caused by curcumin/thapsigargin leads to an influx of calcium into wild-type lysosomes that does not occur in NPC (Figure 3) [6], suggesting that the NPC lysosomal calcium defect may arise from defective calcium entry into the organelle.

Disrupted lysosomal calcium uptake in NPC1

Figure 3
Disrupted lysosomal calcium uptake in NPC1

Under normal conditions, we have discovered that lysosomes mediate the uptake of calcium from the cytosol following transient elevation (A), e.g. during calcium release from the ER induced by thapsigargin (via inhibition of SERCA). In NPC1 patient fibroblasts, there is a defect in calcium uptake into lysosomes following thapsigargin treatment (A). This indicates that the lysosomal calcium defect observed in NPC cells is the result of inhibition of an as yet unidentified lysosomal calcium transporter (B). Reproduced from [6] with permission.

Figure 3
Disrupted lysosomal calcium uptake in NPC1

Under normal conditions, we have discovered that lysosomes mediate the uptake of calcium from the cytosol following transient elevation (A), e.g. during calcium release from the ER induced by thapsigargin (via inhibition of SERCA). In NPC1 patient fibroblasts, there is a defect in calcium uptake into lysosomes following thapsigargin treatment (A). This indicates that the lysosomal calcium defect observed in NPC cells is the result of inhibition of an as yet unidentified lysosomal calcium transporter (B). Reproduced from [6] with permission.

Mucolipidosis type IV

MLIV is a rare neurodegenerative LSD caused by mutations in TRPML1, a member of the mucolipin family of predominantly endolysosomal multi-ion transporters [31]. The disease is characterized by progressive early decline associated with increased blood gastrin levels and neuronal loss followed by stabilization for several decades [31]. The TRPML1 protein is a lysosomal limiting membrane protein permeable to multiple ions and is believed to be essential for lysosome biogenesis, although its exact function is unknown [31]. TRPML1-null cells accumulate a number of macromolecules, including sphingolipids, phospholipids, glycosaminoglycans and autofluorescent lipofuscin [31]. Endocytic trafficking defects are apparent, and a potential defect in recycling of lipids caused by enhanced late-endosome–lysosome fusion may underlie the diverse storage material that accumulates in MLIV [31]. Owing to the defects in late-endosome–lysosome reformation and lysosomal biogenesis [4446], it has been suggested that TRPML1 may function as a lysosomal calcium channel regulating fission of the fused heterotypic (late endosomes/lysosomes) and homotypic (late endosomes/late endosomes) organelles [45]. At present, there is no evidence to support this hypothesis, and others have suggested that TRPML1 may, in fact, function predominantly as an iron or proton transporter [29,47,48]. Different studies have provided different pH recordings of the endolysosomal system in MLIV with estimates of increased, decreased or no change in pH [27,48,49]. These different estimates are speculated to be the result of measurement of different compartments, the lysosome in normal cells and the fused intermediary late-endosome–lysosome in TRPML1-null cells, which may, under normal circumstances, have altered pH [31]. It is clear that further work is required to elucidate the exact function of TRPML1.

CHS (Chediak–Higashi syndrome)

CHS is a rare multi-system disease caused by mutations in the lysosomal trafficking regulator LYST/CHS1 [50]. The disease is associated with decreased pigmentation, decreased platelet dense bodies, defective clearance of infectious agents and defective secretion of acidic secretory granules by T-cells [50,51]. Should patients survive into adulthood, they develop neurological impairment, including neuropathy, cerebellar degeneration and seizures [50,51]. Multiple enlarged lysosomes are seen by electron microscopy [51] and lysosomal exocytosis is impaired, leading to defects in wound healing [52]. Interestingly, a study of neutrophils isolated from the beige mouse model of CHS indicated that these cells had enhanced calcium uptake into lysosomes [53]. The number of ATP-dependent calcium-uptake pumps and their affinity for calcium was enhanced, suggesting that CHS lysosomes may be overefficient at resequestering calcium, which may in turn alter local calcium signalling events [53]. It is therefore possible that several of the defects observed in CHS may be associated with lower than normal concentration of local extravesicular lysosomal calcium (potentially below the 0.1–1 μM calcium necessary for fusion), perturbing lysosome-related organelle biogenesis.

Mucopolysaccharidosis type 1

A recent study has described the presence of a lysosomal calcium defect in mucopolysaccharidosis type 1 mouse cells [54]. Elevated lysosomal calcium levels were reported in these cells [54]. Interestingly, this defect was coupled with an increase in lysosomal pH [54]. These data appear to conflict with the findings of others that lysosomal calcium store entry requires normal lysosomal proton content with de-acidification of the store, leading to the loss of calcium [5]. The use of low-affinity calcium-binding dextran to directly measure intralysosomal calcium would prove useful in confirming these findings.

Conclusions

The lysosome has now emerged as an important calcium store that is involved in fundamental cellular processes, including regulation of endocytosis, recycling of macromolecules and intracellular signalling. With emerging advancements in the tools with which we can study calcium homoeostasis in this compartment [5,6,55,56], further developments are likely in the near future. The discoveries that defective lysosomal calcium homoeostasis underlie the pathogenesis of a number of human diseases may pave the way for future therapeutic intervention.

Lysosomes in Health and Disease: A Biochemical Society Focused Meeting held at Charles Darwin House, London, U.K., 13–14 May 2010. Organized and Edited by Frances Platt (Oxford, U.K.) and Paul Pryor (York, U.K.).

Abbreviations

     
  • AM

    acetoxymethyl ester

  •  
  • BAPTA

    1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid

  •  
  • CHS

    Chediak–Higashi syndrome

  •  
  • ER

    endoplasmic reticulum

  •  
  • LSD

    lysosomal storage disease

  •  
  • MLIV

    mucolipidosis IV

  •  
  • NAADP

    nicotinic acid–adenine dinucleotide phosphate

  •  
  • NPC

    Niemann–Pick type C

  •  
  • SERCA

    sarcoplasmic/endoplasmic reticulum Ca2+-ATPase

  •  
  • TPC

    two-pore channel

  •  
  • TRPML

    transient receptor potential mucolipin

We thank Ian Williams, Brian Tan and Paul Fineran for insightful comments during the writing of the paper.

Funding

K.P. is supported by Action Medical Research. E.L.E. is supported by SPARKS and Newlife UK.

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