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Regulation of TRPML1 function Helen Waller-Evans*1 and Emyr Lloyd-Evans* *School of Biosciences, Sir Martin Evans building, Cardiff University, Museum Avenue, Cardiff, CF15 8AZ, U.K.
Abstract TRPML1 is a ubiquitously expressed cation channel found on lysosomes and late endosomes. Mutations in TRPML1 cause mucolipidosis type IV and it has been implicated in Alzheimer’s disease and HIV. However, the mechanisms by which TRPML1 activity is regulated are not well understood. This review summarizes the current understanding of TRPML1 activation and regulation.
Introduction Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) is a cation channel found in late endosomes and lysosomes whose activity is vital for proper functioning of these compartments [1]. This review will focus on TRPML1 ion selectivity and the mechanisms by which its activity is regulated. TRPML1, or mucolipin 1, is a ubiquitously expressed late-endosomal/lysosomal protein encoded by the gene MCOLN1 [2–4]. Mutations in MCOLN1 lead to mucolipidosis type IV (MLIV), a neurodegenerative lysosomal storage disorder with psychomotor retardation and visual impairment as the major symptoms [5]. At the cellular level, TRPML1 null cells are characterized by endocytic mistrafficking defects and intralysosomal accumulation of lipofuscin and other macromolecules including phospholipids, gangliosides and mucopolysaccharides [6,7]. TRPML1 is a 6 transmembrane domain protein closely related to two other mucolipins, TRPML2 and TRPML3, and part of the wider transient receptor potential (TRP) channel superfamily. TRPMLs differ from classical TRP channels in that they contain a large cytosolic (or luminal) loop between membrane domains one and two, and have unusually short C-terminal tails [8]. TRPMLs are conserved in Drosophila melanogaster and Caenorhabditis elegans, with each having one TRPML gene, trpml and cup5 respectively. Mutations in trpml recapitulate all major phenotypes of MLIV including locomotor and retinal defects, and intralysosomal accumulation of lipofuscin [9]. Mutations in cup-5 lead to an increased number of lysosomes and endocytic trafficking defects causing impaired lysosomal function and embryonic lethality [10,11].
TRPML1 cation selectivity TRPML1 is long established as being permeable to multiple cations, including Ca2 + . LaPlante et al. injected RNA for Key words: ion selectivity, pH regulation, PtdIns(3,5)P2 , mucolipin 1, TRPML1. Abbreviations: MLIV, mucolipidosis type IV; MLSA1, mucolipin synthetic agonist 1; NAADP, nicotinic acid adenine dinucleotide phosphate; TPC, two-pore channel; TRP, transient receptor potential. 1 To whom correspondence should be addressed (email [email protected]).
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human TRPML1 into Xenopus laevis oocytes and recorded channel opening and conductance in response to various ions by patch-clamping across the plasma membrane. Currents across TRPML1 were found in response to Ca2 + , K + and Na + , and the open probability is increased by increasing cytosolic Ca2 + [12,13]. Permeability of TRPML1 to Ca2 + , K + and Na + was also found in an in vitro system where TRPML1 was reconstituted into planar lipid bilayers [14]. The Ca2 + permeability of TRPML1 was confirmed by Shen et al. [15] who showed by patch-clamping of vacuolin swollen lysosomes that Ca2 + currents through TRPML1 are increased in response to the synthetic TRPML channel agonist mucolipin synthetic agonist 1 (MLSA1) (discussed in the ‘TRPML1 agonists’ section). However, it should be noted that vacuolin causes all parts of the endocytic system to swell, with the biggest effect on early endosomes, where TRPML3 is located, so it is possible that recordings were made through TRPML3 [16,17]. TRPML1 has been found to be permeable to divalent cations other than Ca2 + . Dong et al. [18] showed that TRPML1 is permeable to Fe2 + but not Fe3 + . The authors overexpressed a constitutively active form of TRPML1 in HEK293T cells, which causes some TRPML1 to be mislocalized to the plasma membrane where it can be patch-clamped. In order to recapitulate physiological lateendosomal/lysosomal conditions, cells were bathed in an extracellular fluid at pH 4.6. Inwardly rectifying current was observed in response to extracellular addition of Fe2 + but not Fe3 + or N-methyl-D-glucamine, indicating that TRPML1 is selectively permeable to divalent Fe ions. This is supported by the finding that TRPML1 null cells have lower levels of cytosolic chelatable Fe, indicating that Fe ions brought in by endocytosis are not escaping the endocytic system. Dong et al. [18] also found that TRPML1 is permeable to Zn2 + . Altered Zn2 + homoeostasis has been found in TRPML1 null cells. Silencing of TRPML1 by shRNA in HEK293T cells leads to an increase in total chelatable Zn levels, which was localized to enlarged lysosomes formed following shRNA treatment [19]. However, this only occurs when high levels of Zn2 + are added to the cell, so it is not clear whether this true in physiological conditions. Biochem. Soc. Trans. (2015) 43, 442–446; doi:10.1042/BST20140311
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TRPML1 has been found to be permeable to Ca2 + , K + , Na + , Fe2 + and Zn2 + , with the current indicating that the ions would enter the cytosol from the lysosomal/late-endosomal lumen. However, Kiselyov et al. [1] have suggested that TRPML1 acts as a H + channel, with protons moving into the lysosomal lumen from the cytoplasm. It is important to remember when assessing the evidence for the cation permeability of TRPML1 that experiments have utilized overexpression, reconstitution of channels into bilayers and swelling of endocytic compartments using vacuolin. That is, they have not been conducted under physiological conditions and may not reflect the situation in unaltered cells. In addition, several findings such as permeability to Fe2 + , Zn2 + and H + have not been replicated. This means that the true extent of TRPML1 cation permeability is uncertain although as permeability to Ca2 + has been replicated by several different groups using different experimental systems, it is relatively safe to believe that TRPML1 is permeable to Ca2 + .
TRPML1 agonists Phosphoinositol (3,5)-bisphosphate (PI(3,5)P2 ) PI(3,5)P2 is a phosphoiniositol found at low levels within cells which is predominantly located in late endosomes and lysosomes in mammalian and insect cells and in the vacuole in yeast [20]. Mutations in Fab1/PIKfyve, the enzyme responsible for PI(3,5)P2 production in Drosophila or overexpression of dominant-negative Fab1/PIKfyve in mammalian cells lead to an accumulation of swollen endosomes within cells [21,22]. This is also the case in mammalian cells treated with a small molecule inhibitor of PI(3,5)P2 synthesis, YM201636, where treatment resulted in the accumulation of large vesicles positive for both late endosomal and lysosomal markers and impaired transport of retroviruses out of late endosomes [23]. The accumulation of late endosomes and lysosomes and defect in endocytic trafficking are similar to these seen in TRPML1 null cells [24]. Upon observing these similarities, Dong et al. [24] investigated whether PI(3,5)P2 may be an endogenous ligand for TRPML1. Water soluble diC8 PI(3,5)P2 was added to mammalian cells overexpressing TRPML1. TRPML1-dependent currents in vacuolin swollen late endosomes/lysosomes increased approximately 18-fold compared with basal activity, indicating that PI(3,5)P2 can indeed activate TRPML1. This effect was not seen in a mutant channel locked in an open state, suggesting that PI(3,5)P2 increases the open probability of TRPML1. This was proposed to occur by direct binding of PI(3,5)P2 to the N-terminus of TRPML1, which was shown in vitro. The authors suggest that TRPML1 is activated by PI(3,5)P2 to promote correct fusion/fission in the endocytic system and that the loss of this process is the cause of the accumulation of swollen lysosomes in PI(3,5)P2 depleted cells.
Drosophila trpml has also been found to be sensitive to PI(3,5)P2 [25]. trpml was expressed in HEK293T cells, and found to be localized to endosomes and lysosomes. Patch-clamping of vacuolin swollen lysosomes in these cells revealed that current increased upon addition of PI(3,5)P2 in a dose-dependent manner. This study also confirmed that Drosophila trpml has a similar function to mammalian TRPML1 by rescuing the pupal lethality phenotype seen in null flies using expression of human TRPML1. Taken together, these experiments provide very strong evidence for PI(3,5)P2 activation of TRPML1.
Nicotinic acid adenine dinucleotide phosphate (NAADP) Zhang et al. [26] suggested that TRPML1 releases Ca2 + in response to the potent Ca2 + releasing second messenger NAADP. The evidence in favour of this is that endothelin (an extracellular signal known to trigger NAADP signalling [27]) induced Ca2 + release is reduced in cells where TRPML1 has been silenced. However, no Western blot for TRPML1 is shown for the silenced cells, so it is impossible to gauge the extent of any knockdown. The authors also found that anti-TRPML1 antibodies could eliminate NAADP-mediated Ca2 + release from purified lysosomes reconstituted into lipid bilayers, although again, no evidence of TRPML1 removal is shown. In a later study, Zhang et al. [28] showed that purified lysosomes reconstituted into lipid bilayers taken from MLIV patient fibroblasts (TRPML1 null) do not release Ca2 + in response to NAADP, and that this defect can be rescued by re-expression of TRPML1. The authors also found that there is less fusion between late endosomes and lysosomes in MLIV patient fibroblasts, but that this trafficking defect can also be corrected by re-expression of TRPML1 and addition of NAADP suggesting a role for NAADP-mediated signalling in endocytic trafficking, which has also been suggested by others [29]. As of yet, no other group has published confirmation of this work, whereas several groups have found evidence that a two-pore channel (TPC), TPC2, is the NAADP sensitive channel of the lysosome [30–33]. In fact, the group of Muallem have data that demonstrate that TRPML1 is not NAADP sensitive. Yamaguchi et al. [33] found that overexpression of TRPML1 did not increase NAADP-mediated Ca2 + release in intact cells, whereas overexpression of TPC2 did suggesting that TPC2, not TRPML1, is responsible for NAADP-mediated Ca2 + release. The authors also found that TRPML1 and TPC2 associate on the lysosomal membrane and co-immunoprecipitate. It is therefore possible that by using an antibody to remove TRPML1 from lysosomal membranes, Zhang et al. [26] also removed TPC2 which would explain the loss of NAADP-mediated Ca2 + signalling in these preparations. In conclusion, the weight of evidence suggests that TRPML1 is not sensitive to NAADP.
Mucolipin synthetic agonist 1 (MLSA1) MLSA1 was discovered by Shen et al. [15] in a low throughput screen for TRPML1 activators. It is chemically closely C The
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related to SF-51, a small molecule activator of TRPML3 identified by Grimm et al. [34]. This was found to also weakly activate TRPML1 and was used as the basis for the screen. MLSA1 induces the same level of inwardly rectifying Ca2 + current as the endogenous ligand PI(3,5)P2 when used at 10 μM. It also activates TRPML2 and TRPML3. Shen et al. [15] circumvented this potential problem by performing single channel recordings from endo-lysosomes swollen with vacuolin, where TRPML1 is the major TRPML present. However, as mentioned earlier, vacuolin causes all endocytic compartments to swell meaning that recording may have been from TRPML3 [16,17]. It has recently been discovered that MLSA1 alone cannot activate Drosophila trpml [35]. Rather, it acts to increase the sensitivity of trpml to the endogenous ligand PI(3,5)P2 . This is in contrast to the authors findings that mouse TRPML1 can be activated by MLSA1 even in the absence of PI(3,5)P2 . The need for PI(3,5)P2 for activation of Drosophila trpml is abolished by introduction of mutations that mimic those found in the varitint-waddler mouse, which cause TRPML3 to become constitutively active. The authors suggest that MLSA1 works on Drosophila trpml by stabilizing the open conformation of the channel whereas it is able to induce channel opening in mammalian TRPML1.
Regulation of TRPML1 Regulation by pH Both the ion conductance and channel open probability of TRPML1 are regulated by pH. Xu et al. [36] found that channel conductance in a constitutively active form of TRPML1 is higher at pH 4.6 than at pH 7.4. This conductance was abolished in the absence of Ca2 + and Na + . This indicates that TRPML1 is more permeable to Ca2 + ions at acidic pH, which would fit with its localization on late endosomes and lysosomes. This increased conductance at acidic pH was also found to be true for a constitutively active mutant of Drosophila trpml. Feng et al. [25] tested conductance of constitutively active forms of trpml and human TRPML1 in a pH range of 3–8 and found that pH-dependent conductance follows a bell-shaped curve with human TRPML1 equally permeable at pH 4.0 and 5.0, and Drosophila trpml most permeable at pH 5.0. The increased channel conductance at acidic pH was confirmed by Chen et al. [37] using wild-type TRPML1 and F408, an MLIV causing TRPML1 mutant missing amino acid 408. These channels are not constitutively active but were activated by PI(3,5)P2 , SF-22 (a small molecule agonist identified by Grimm et al. [34]) or the novel small molecule TRPML1 agonist MK6-83. Upon channel activation, channel conductance was higher at pH 4.6 than at pH 7.2. Interestingly, the increase in conductance induced by PI(3,5)P2 was much reduced in the F408 mutant compared with wild-type, which could be the mechanism by which it causes disease. C The
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In work that seems to contradict that of Xu, Feng and Chen [35–37], Raychowdhury et al. [14] found that TRPML1 activity is inhibited by acidic pH. The authors reconstituted endosomes expressing wild-type, F408 or V446L mutants into lipid bilayers and measured conductance across single channels. They found that decreasing the pH to 5.0 reduced mean current in wild-type channels but not F408 or V446L mutants. However, they go on to show that pH does not affect single channel conductance of TRPML1. Rather, their traces indicate that reducing pH reduced the open probability of TRPML1 in the absence of any endogenous ligand or constitutively activating mutation. The two sets of data can therefore be reconciled; TRPML1 is more likely to open as pH rises, but when it is open, ion conductance is higher at more acidic pH. However, as is the case for ion conductance, it should be noted that most of this work has been conducted in non-physiological conditions, using constitutively active mutants, vacuolin swollen endocytic compartments and receptors in the absence of ligand. The effect of pH on TRPML1 function has therefore not been fully elucidated.
Inhibition of TRPML1 It has been suggested that TRPML1 or Drosophila trpml is inhibited by sphingomyelin [15], PI(4,5)P2 [24,25] and trivalent cations [25]. Shen et al. [15] found that SF-51induced activation of TRPML1 is reduced in the presence of sphingomyelin. Dong et al. [24] found that PI(3,5)P2 - and SF-51-induced TRPML1 activity is reduced in the presence of PI(4,5)P2 in inside-out cell recordings of TRPML1 overexpressing cells. This has been confirmed in Drosophila trpml where PI(4,5)P2 inhibited PI(3,5)P2 -induced trpml activity. It has been suggested that PI(4,5)P2 may inhibit TRPML1 function at the plasma membrane although it is being trafficked to late-endosomes and lysosomes, where it can be activated by PI(3,5)P2 [25]. Feng et al. [25] also found that constitutively active forms of both Drosophila trpml and human TRPML1 are inhibited by La3 + and Gd3 + and that trpml is inhibited by Fe3 + . However, apart from the finding that TRPML1 is inhibited by PI(4,5)P2 , none of these findings have been replicated and the mechanisms of TRPML1 inhibition remain largely unknown.
TRPML1 in disease In addition to MLIV, TRPML1 has been implicated in other diseases. Shen et al. [15] suggested TRPML1 involvement in the rare neurodegenerative lysosomal storage disorder Niemann–Pick type C, caused by defects in the NiemannPick disease, type C1 (NPC1) protein. It was found that lysosomal Ca2 + release in response to MLSA1 is reduced in NPC1 null cells and this was proposed to be due to inhibition of TRPML1 by sphingomyelin, which accumulates in lysosomes in NPC1 null cells. This was also found to be the case for the primary sphingomyelin storage disorder Niemann–Pick type A. However, the authors found no difference in lysosomal Ca2 + content between wild-type and
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NPC1 null cells, despite this being widely reported [38–41]. The authors found that treating NPC1 null cells with MLSA1 led to an improvement in phenotypes. However, this could be due to a rise in cytoplasmic Ca2 + content overcoming a block in late-endosome/lysosome fusion, as suggested by Lloyd-Evans et al. [38]. More recently, TRPML1 involvement has been proposed to contribute to Aβ peptide accumulation in HIV patients. Expression of the HIV coat protein gp120 in an APP/PS1 mouse model of early onset Alzheimer’s disease accelerates the accumulation of Aβ [42]. Treatment of primary neurons with gp120 also induces accumulation of Aβ and sphingomyelin, which was thought to be inhibiting TRPML1 and preventing clearance of Aβ. This is in contrast with the situation in Alzheimer’s disease, where sphingomyelin levels are reduced [43]. Ca2 + release in response to MLSA1 was enhanced in cells treated with gp120, which contradicts the idea that TRPML1 is inhibited by sphingomyelin. This was followed by clearance of Aβ and sphingomyelin. However, the authors do not show direct involvement of TRPML1 or whether TRPML1 expression or activity is altered in HIV. These data indicate the possible involvement of lysosomal Ca2 + in HIV-induced Aβ accumulation and possibly also in Alzheimer’s disease.
Conclusions There is currently a lot of contradictory evidence regarding the activation, regulation and role of TRPML1. There is good evidence that TRPML1 is permeable to Ca2 + ions, that it is activated by PI(3,5)P2 and that its activity is regulated by pH. It is also clear that correct TRPML1 function is vital for proper cell function. However, much about the protein and how its activity is regulated remains unknown and it is clear that more work is needed to fully elucidate the function of TRPML1.
Funding This work was supported by the Action Medical Research (to H.W.E.); the Henry Smith charity (to H.W.-E.); the Research Councils UK fellowship (to E.L.-E.); the Royal Society research grant (to E.L.-E.); and the March of Dimes Basil O’Connor starter scholar research award (to E.L.-E.).
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34 Grimm, C., Jors, S., Saldanha, S.A., Obukhov, A.G., Pan, B., Oshima, K., Cuajungco, M.P., Chase, P., Hodder, P. and Heller, S. (2010) Small molecule activators of TRPML3. Chem. Biol. 17, 135–148 CrossRef PubMed 35 Feng, X., Xiong, J., Lu, Y., Xia, X. and Zhu, M.X. (2014) Differential mechanisms of action of the mucolipin synthetic agonist, ML-SA1, on insect TRPML and mammalian TRPML1. Cell Calcium 56, 446–456 CrossRef 36 Xu, H., Delling, M., Li, L., Dong, X. and Clapham, D.E. (2007) Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice. Proc. Natl. Acad. Sci. U. S. A. 104, 18321–18326 CrossRef PubMed 37 Chen, C.C., Keller, M., Hess, M., Schiffmann, R., Urban, N., Wolfgardt, A., Schaefer, M., Bracher, F., Biel, M., Wahl-Schott, C. and Grimm, C. (2014) A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV. Nat. Commun. 5, 4681 PubMed 38 Lloyd-Evans, E., Morgan, A.J., He, X., Smith, D.A., Elliot-Smith, E., Sillence, D.J., Churchill, G.C., Schuchman, E.H., Galione, A. and Platt, F.M. (2008) Niemann–Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nat. Med. 14, 1247–1255 CrossRef PubMed 39 Lee, H., Lee, J.K., Min, W.K., Bae, J.H., He, X., Schuchman, E.H., Bae, J.S. and Jin, H.K. (2010) Bone marrow-derived mesenchymal stem cells prevent the loss of Niemann–Pick type C mouse Purkinje neurons by correcting sphingolipid metabolism and increasing sphingosine-1-phosphate. Stem Cells 28, 821–831 CrossRef PubMed 40 Xu, M., Liu, K., Swaroop, M., Porter, F.D., Sidhu, R., Firnkes, S., Ory, D.S., Marugan, J.J., Xiao, J., Southall, N. et al. (2012) delta-Tocopherol reduces lipid accumulation in Niemann–Pick type C1 and Wolman cholesterol storage disorders. J. Biol. Chem. 287, 39349–39360 CrossRef PubMed 41 Visentin, S., De Nuccio, C., Bernardo, A., Pepponi, R., Ferrante, A., Minghetti, L. and Popoli, P. (2013) The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann–Pick type C patients. J. Neurosci. 33, 15388–15393 CrossRef PubMed 42 Bae, M., Patel, N., Xu, H., Lee, M., Tominaga-Yamanaka, K., Nath, A., Geiger, J., Gorospe, M., Mattson, M.P. and Haughey, N.J. (2014) Activation of TRPML1 clears intraneuronal Abeta in preclinical models of HIV infection. J. Neurosci. 34, 11485–11503 CrossRef PubMed 43 He, X., Huang, Y., Li, B., Gong, C.X. and Schuchman, E.H. (2010) Deregulation of sphingolipid metabolism in Alzheimer’s disease. Neurobiol. Aging 31, 398–408 CrossRef PubMed Received 18 December 2014 doi:10.1042/BST20140311
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Regulation of TRPML1 function Helen Waller-Evans*1 and Emyr Lloyd-Evans* *School of Biosciences, Sir Martin Evans building, Cardiff University, Museum Avenue, Cardiff, CF15 8AZ, U.K.
Abstract TRPML1 is a ubiquitously expressed cation channel found on lysosomes and late endosomes. Mutations in TRPML1 cause mucolipidosis type IV and it has been implicated in Alzheimer’s disease and HIV. However, the mechanisms by which TRPML1 activity is regulated are not well understood. This review summarizes the current understanding of TRPML1 activation and regulation.
Introduction Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) is a cation channel found in late endosomes and lysosomes whose activity is vital for proper functioning of these compartments [1]. This review will focus on TRPML1 ion selectivity and the mechanisms by which its activity is regulated. TRPML1, or mucolipin 1, is a ubiquitously expressed late-endosomal/lysosomal protein encoded by the gene MCOLN1 [2–4]. Mutations in MCOLN1 lead to mucolipidosis type IV (MLIV), a neurodegenerative lysosomal storage disorder with psychomotor retardation and visual impairment as the major symptoms [5]. At the cellular level, TRPML1 null cells are characterized by endocytic mistrafficking defects and intralysosomal accumulation of lipofuscin and other macromolecules including phospholipids, gangliosides and mucopolysaccharides [6,7]. TRPML1 is a 6 transmembrane domain protein closely related to two other mucolipins, TRPML2 and TRPML3, and part of the wider transient receptor potential (TRP) channel superfamily. TRPMLs differ from classical TRP channels in that they contain a large cytosolic (or luminal) loop between membrane domains one and two, and have unusually short C-terminal tails [8]. TRPMLs are conserved in Drosophila melanogaster and Caenorhabditis elegans, with each having one TRPML gene, trpml and cup5 respectively. Mutations in trpml recapitulate all major phenotypes of MLIV including locomotor and retinal defects, and intralysosomal accumulation of lipofuscin [9]. Mutations in cup-5 lead to an increased number of lysosomes and endocytic trafficking defects causing impaired lysosomal function and embryonic lethality [10,11].
TRPML1 cation selectivity TRPML1 is long established as being permeable to multiple cations, including Ca2 + . LaPlante et al. injected RNA for Key words: ion selectivity, pH regulation, PtdIns(3,5)P2 , mucolipin 1, TRPML1. Abbreviations: MLIV, mucolipidosis type IV; MLSA1, mucolipin synthetic agonist 1; NAADP, nicotinic acid adenine dinucleotide phosphate; TPC, two-pore channel; TRP, transient receptor potential. 1 To whom correspondence should be addressed (email [email protected]).
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human TRPML1 into Xenopus laevis oocytes and recorded channel opening and conductance in response to various ions by patch-clamping across the plasma membrane. Currents across TRPML1 were found in response to Ca2 + , K + and Na + , and the open probability is increased by increasing cytosolic Ca2 + [12,13]. Permeability of TRPML1 to Ca2 + , K + and Na + was also found in an in vitro system where TRPML1 was reconstituted into planar lipid bilayers [14]. The Ca2 + permeability of TRPML1 was confirmed by Shen et al. [15] who showed by patch-clamping of vacuolin swollen lysosomes that Ca2 + currents through TRPML1 are increased in response to the synthetic TRPML channel agonist mucolipin synthetic agonist 1 (MLSA1) (discussed in the ‘TRPML1 agonists’ section). However, it should be noted that vacuolin causes all parts of the endocytic system to swell, with the biggest effect on early endosomes, where TRPML3 is located, so it is possible that recordings were made through TRPML3 [16,17]. TRPML1 has been found to be permeable to divalent cations other than Ca2 + . Dong et al. [18] showed that TRPML1 is permeable to Fe2 + but not Fe3 + . The authors overexpressed a constitutively active form of TRPML1 in HEK293T cells, which causes some TRPML1 to be mislocalized to the plasma membrane where it can be patch-clamped. In order to recapitulate physiological lateendosomal/lysosomal conditions, cells were bathed in an extracellular fluid at pH 4.6. Inwardly rectifying current was observed in response to extracellular addition of Fe2 + but not Fe3 + or N-methyl-D-glucamine, indicating that TRPML1 is selectively permeable to divalent Fe ions. This is supported by the finding that TRPML1 null cells have lower levels of cytosolic chelatable Fe, indicating that Fe ions brought in by endocytosis are not escaping the endocytic system. Dong et al. [18] also found that TRPML1 is permeable to Zn2 + . Altered Zn2 + homoeostasis has been found in TRPML1 null cells. Silencing of TRPML1 by shRNA in HEK293T cells leads to an increase in total chelatable Zn levels, which was localized to enlarged lysosomes formed following shRNA treatment [19]. However, this only occurs when high levels of Zn2 + are added to the cell, so it is not clear whether this true in physiological conditions. Biochem. Soc. Trans. (2015) 43, 442–446; doi:10.1042/BST20140311
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TRPML1 has been found to be permeable to Ca2 + , K + , Na + , Fe2 + and Zn2 + , with the current indicating that the ions would enter the cytosol from the lysosomal/late-endosomal lumen. However, Kiselyov et al. [1] have suggested that TRPML1 acts as a H + channel, with protons moving into the lysosomal lumen from the cytoplasm. It is important to remember when assessing the evidence for the cation permeability of TRPML1 that experiments have utilized overexpression, reconstitution of channels into bilayers and swelling of endocytic compartments using vacuolin. That is, they have not been conducted under physiological conditions and may not reflect the situation in unaltered cells. In addition, several findings such as permeability to Fe2 + , Zn2 + and H + have not been replicated. This means that the true extent of TRPML1 cation permeability is uncertain although as permeability to Ca2 + has been replicated by several different groups using different experimental systems, it is relatively safe to believe that TRPML1 is permeable to Ca2 + .
TRPML1 agonists Phosphoinositol (3,5)-bisphosphate (PI(3,5)P2 ) PI(3,5)P2 is a phosphoiniositol found at low levels within cells which is predominantly located in late endosomes and lysosomes in mammalian and insect cells and in the vacuole in yeast [20]. Mutations in Fab1/PIKfyve, the enzyme responsible for PI(3,5)P2 production in Drosophila or overexpression of dominant-negative Fab1/PIKfyve in mammalian cells lead to an accumulation of swollen endosomes within cells [21,22]. This is also the case in mammalian cells treated with a small molecule inhibitor of PI(3,5)P2 synthesis, YM201636, where treatment resulted in the accumulation of large vesicles positive for both late endosomal and lysosomal markers and impaired transport of retroviruses out of late endosomes [23]. The accumulation of late endosomes and lysosomes and defect in endocytic trafficking are similar to these seen in TRPML1 null cells [24]. Upon observing these similarities, Dong et al. [24] investigated whether PI(3,5)P2 may be an endogenous ligand for TRPML1. Water soluble diC8 PI(3,5)P2 was added to mammalian cells overexpressing TRPML1. TRPML1-dependent currents in vacuolin swollen late endosomes/lysosomes increased approximately 18-fold compared with basal activity, indicating that PI(3,5)P2 can indeed activate TRPML1. This effect was not seen in a mutant channel locked in an open state, suggesting that PI(3,5)P2 increases the open probability of TRPML1. This was proposed to occur by direct binding of PI(3,5)P2 to the N-terminus of TRPML1, which was shown in vitro. The authors suggest that TRPML1 is activated by PI(3,5)P2 to promote correct fusion/fission in the endocytic system and that the loss of this process is the cause of the accumulation of swollen lysosomes in PI(3,5)P2 depleted cells.
Drosophila trpml has also been found to be sensitive to PI(3,5)P2 [25]. trpml was expressed in HEK293T cells, and found to be localized to endosomes and lysosomes. Patch-clamping of vacuolin swollen lysosomes in these cells revealed that current increased upon addition of PI(3,5)P2 in a dose-dependent manner. This study also confirmed that Drosophila trpml has a similar function to mammalian TRPML1 by rescuing the pupal lethality phenotype seen in null flies using expression of human TRPML1. Taken together, these experiments provide very strong evidence for PI(3,5)P2 activation of TRPML1.
Nicotinic acid adenine dinucleotide phosphate (NAADP) Zhang et al. [26] suggested that TRPML1 releases Ca2 + in response to the potent Ca2 + releasing second messenger NAADP. The evidence in favour of this is that endothelin (an extracellular signal known to trigger NAADP signalling [27]) induced Ca2 + release is reduced in cells where TRPML1 has been silenced. However, no Western blot for TRPML1 is shown for the silenced cells, so it is impossible to gauge the extent of any knockdown. The authors also found that anti-TRPML1 antibodies could eliminate NAADP-mediated Ca2 + release from purified lysosomes reconstituted into lipid bilayers, although again, no evidence of TRPML1 removal is shown. In a later study, Zhang et al. [28] showed that purified lysosomes reconstituted into lipid bilayers taken from MLIV patient fibroblasts (TRPML1 null) do not release Ca2 + in response to NAADP, and that this defect can be rescued by re-expression of TRPML1. The authors also found that there is less fusion between late endosomes and lysosomes in MLIV patient fibroblasts, but that this trafficking defect can also be corrected by re-expression of TRPML1 and addition of NAADP suggesting a role for NAADP-mediated signalling in endocytic trafficking, which has also been suggested by others [29]. As of yet, no other group has published confirmation of this work, whereas several groups have found evidence that a two-pore channel (TPC), TPC2, is the NAADP sensitive channel of the lysosome [30–33]. In fact, the group of Muallem have data that demonstrate that TRPML1 is not NAADP sensitive. Yamaguchi et al. [33] found that overexpression of TRPML1 did not increase NAADP-mediated Ca2 + release in intact cells, whereas overexpression of TPC2 did suggesting that TPC2, not TRPML1, is responsible for NAADP-mediated Ca2 + release. The authors also found that TRPML1 and TPC2 associate on the lysosomal membrane and co-immunoprecipitate. It is therefore possible that by using an antibody to remove TRPML1 from lysosomal membranes, Zhang et al. [26] also removed TPC2 which would explain the loss of NAADP-mediated Ca2 + signalling in these preparations. In conclusion, the weight of evidence suggests that TRPML1 is not sensitive to NAADP.
Mucolipin synthetic agonist 1 (MLSA1) MLSA1 was discovered by Shen et al. [15] in a low throughput screen for TRPML1 activators. It is chemically closely C The
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related to SF-51, a small molecule activator of TRPML3 identified by Grimm et al. [34]. This was found to also weakly activate TRPML1 and was used as the basis for the screen. MLSA1 induces the same level of inwardly rectifying Ca2 + current as the endogenous ligand PI(3,5)P2 when used at 10 μM. It also activates TRPML2 and TRPML3. Shen et al. [15] circumvented this potential problem by performing single channel recordings from endo-lysosomes swollen with vacuolin, where TRPML1 is the major TRPML present. However, as mentioned earlier, vacuolin causes all endocytic compartments to swell meaning that recording may have been from TRPML3 [16,17]. It has recently been discovered that MLSA1 alone cannot activate Drosophila trpml [35]. Rather, it acts to increase the sensitivity of trpml to the endogenous ligand PI(3,5)P2 . This is in contrast to the authors findings that mouse TRPML1 can be activated by MLSA1 even in the absence of PI(3,5)P2 . The need for PI(3,5)P2 for activation of Drosophila trpml is abolished by introduction of mutations that mimic those found in the varitint-waddler mouse, which cause TRPML3 to become constitutively active. The authors suggest that MLSA1 works on Drosophila trpml by stabilizing the open conformation of the channel whereas it is able to induce channel opening in mammalian TRPML1.
Regulation of TRPML1 Regulation by pH Both the ion conductance and channel open probability of TRPML1 are regulated by pH. Xu et al. [36] found that channel conductance in a constitutively active form of TRPML1 is higher at pH 4.6 than at pH 7.4. This conductance was abolished in the absence of Ca2 + and Na + . This indicates that TRPML1 is more permeable to Ca2 + ions at acidic pH, which would fit with its localization on late endosomes and lysosomes. This increased conductance at acidic pH was also found to be true for a constitutively active mutant of Drosophila trpml. Feng et al. [25] tested conductance of constitutively active forms of trpml and human TRPML1 in a pH range of 3–8 and found that pH-dependent conductance follows a bell-shaped curve with human TRPML1 equally permeable at pH 4.0 and 5.0, and Drosophila trpml most permeable at pH 5.0. The increased channel conductance at acidic pH was confirmed by Chen et al. [37] using wild-type TRPML1 and F408, an MLIV causing TRPML1 mutant missing amino acid 408. These channels are not constitutively active but were activated by PI(3,5)P2 , SF-22 (a small molecule agonist identified by Grimm et al. [34]) or the novel small molecule TRPML1 agonist MK6-83. Upon channel activation, channel conductance was higher at pH 4.6 than at pH 7.2. Interestingly, the increase in conductance induced by PI(3,5)P2 was much reduced in the F408 mutant compared with wild-type, which could be the mechanism by which it causes disease. C The
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In work that seems to contradict that of Xu, Feng and Chen [35–37], Raychowdhury et al. [14] found that TRPML1 activity is inhibited by acidic pH. The authors reconstituted endosomes expressing wild-type, F408 or V446L mutants into lipid bilayers and measured conductance across single channels. They found that decreasing the pH to 5.0 reduced mean current in wild-type channels but not F408 or V446L mutants. However, they go on to show that pH does not affect single channel conductance of TRPML1. Rather, their traces indicate that reducing pH reduced the open probability of TRPML1 in the absence of any endogenous ligand or constitutively activating mutation. The two sets of data can therefore be reconciled; TRPML1 is more likely to open as pH rises, but when it is open, ion conductance is higher at more acidic pH. However, as is the case for ion conductance, it should be noted that most of this work has been conducted in non-physiological conditions, using constitutively active mutants, vacuolin swollen endocytic compartments and receptors in the absence of ligand. The effect of pH on TRPML1 function has therefore not been fully elucidated.
Inhibition of TRPML1 It has been suggested that TRPML1 or Drosophila trpml is inhibited by sphingomyelin [15], PI(4,5)P2 [24,25] and trivalent cations [25]. Shen et al. [15] found that SF-51induced activation of TRPML1 is reduced in the presence of sphingomyelin. Dong et al. [24] found that PI(3,5)P2 - and SF-51-induced TRPML1 activity is reduced in the presence of PI(4,5)P2 in inside-out cell recordings of TRPML1 overexpressing cells. This has been confirmed in Drosophila trpml where PI(4,5)P2 inhibited PI(3,5)P2 -induced trpml activity. It has been suggested that PI(4,5)P2 may inhibit TRPML1 function at the plasma membrane although it is being trafficked to late-endosomes and lysosomes, where it can be activated by PI(3,5)P2 [25]. Feng et al. [25] also found that constitutively active forms of both Drosophila trpml and human TRPML1 are inhibited by La3 + and Gd3 + and that trpml is inhibited by Fe3 + . However, apart from the finding that TRPML1 is inhibited by PI(4,5)P2 , none of these findings have been replicated and the mechanisms of TRPML1 inhibition remain largely unknown.
TRPML1 in disease In addition to MLIV, TRPML1 has been implicated in other diseases. Shen et al. [15] suggested TRPML1 involvement in the rare neurodegenerative lysosomal storage disorder Niemann–Pick type C, caused by defects in the NiemannPick disease, type C1 (NPC1) protein. It was found that lysosomal Ca2 + release in response to MLSA1 is reduced in NPC1 null cells and this was proposed to be due to inhibition of TRPML1 by sphingomyelin, which accumulates in lysosomes in NPC1 null cells. This was also found to be the case for the primary sphingomyelin storage disorder Niemann–Pick type A. However, the authors found no difference in lysosomal Ca2 + content between wild-type and
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NPC1 null cells, despite this being widely reported [38–41]. The authors found that treating NPC1 null cells with MLSA1 led to an improvement in phenotypes. However, this could be due to a rise in cytoplasmic Ca2 + content overcoming a block in late-endosome/lysosome fusion, as suggested by Lloyd-Evans et al. [38]. More recently, TRPML1 involvement has been proposed to contribute to Aβ peptide accumulation in HIV patients. Expression of the HIV coat protein gp120 in an APP/PS1 mouse model of early onset Alzheimer’s disease accelerates the accumulation of Aβ [42]. Treatment of primary neurons with gp120 also induces accumulation of Aβ and sphingomyelin, which was thought to be inhibiting TRPML1 and preventing clearance of Aβ. This is in contrast with the situation in Alzheimer’s disease, where sphingomyelin levels are reduced [43]. Ca2 + release in response to MLSA1 was enhanced in cells treated with gp120, which contradicts the idea that TRPML1 is inhibited by sphingomyelin. This was followed by clearance of Aβ and sphingomyelin. However, the authors do not show direct involvement of TRPML1 or whether TRPML1 expression or activity is altered in HIV. These data indicate the possible involvement of lysosomal Ca2 + in HIV-induced Aβ accumulation and possibly also in Alzheimer’s disease.
Conclusions There is currently a lot of contradictory evidence regarding the activation, regulation and role of TRPML1. There is good evidence that TRPML1 is permeable to Ca2 + ions, that it is activated by PI(3,5)P2 and that its activity is regulated by pH. It is also clear that correct TRPML1 function is vital for proper cell function. However, much about the protein and how its activity is regulated remains unknown and it is clear that more work is needed to fully elucidate the function of TRPML1.
Funding This work was supported by the Action Medical Research (to H.W.E.); the Henry Smith charity (to H.W.-E.); the Research Councils UK fellowship (to E.L.-E.); the Royal Society research grant (to E.L.-E.); and the March of Dimes Basil O’Connor starter scholar research award (to E.L.-E.).
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23 Jefferies, H.B., Cooke, F.T., Jat, P., Boucheron, C., Koizumi, T., Hayakawa, M., Kaizawa, H., Ohishi, T., Workman, P., Waterfield, M.D. and Parker, P.J. (2008) A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding. EMBO Rep. 9, 164–170 CrossRef PubMed 24 Dong, X.P., Shen, D., Wang, X., Dawson, T., Li, X., Zhang, Q., Cheng, X., Zhang, Y., Weisman, L.S., Delling, M. and Xu, H. (2010) PI(3,5)P(2) controls membrane trafficking by direct activation of mucolipin Ca(2 + ) release channels in the endolysosome. Nat. Commun. 1, 38 CrossRef PubMed 25 Feng, X., Huang, Y., Lu, Y., Xiong, J., Wong, C.O., Yang, P., Xia, J., Chen, D., Du, G., Venkatachalam, K. et al. (2014) Drosophila TRPML forms PI(3,5)P2-activated cation channels in both endolysosomes and plasma membrane. J. Biol. Chem. 289, 4262–4272 CrossRef PubMed 26 Zhang, F., Jin, S., Yi, F. and Li, P.L. (2009) TRP-ML1 functions as a lysosomal NAADP-sensitive Ca2 + release channel in coronary arterial myocytes. J. Cell. Mol. Med. 13, 3174–3185 CrossRef PubMed 27 Gambara, G., Billington, R.A., Debidda, M., D’Alessio, A., Palombi, F., Ziparo, E., Genazzani, A.A. and Filippini, A. (2008) NAADP-induced Ca(2 + ) signaling in response to endothelin is via the receptor subtype B and requires the integrity of lipid rafts/caveolae. J. Cell. Physiol. 216, 396–404 CrossRef PubMed 28 Zhang, F., Xu, M., Han, W.Q. and Li, P.L. (2011) Reconstitution of lysosomal NAADP-TRP-ML1 signaling pathway and its function in TRP-ML1( − / − ) cells. Am. J. Physiol. Cell. Physiol. 301, C421–C430 CrossRef PubMed 29 Ruas, M., Rietdorf, K., Arredouani, A., Davis, L.C., Lloyd-Evans, E., Koegel, H., Funnell, T.M., Morgan, A.J., Ward, J.A., Watanabe, K. et al. (2010) Purified TPC isoforms form NAADP receptors with distinct roles for Ca(2 + ) signaling and endolysosomal trafficking. Curr. Biol. 20, 703–709 CrossRef PubMed 30 Calcraft, P.J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., Tang, J., Rietdorf, K., Teboul, L., Chuang, K.T. et al. (2009) NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature 459, 596–600 CrossRef PubMed 31 Brailoiu, E., Churamani, D., Cai, X., Schrlau, M.G., Brailoiu, G.C., Gao, X., Hooper, R., Boulware, M.J., Dun, N.J., Marchant, J.S. and Patel, S. (2009) Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling. J. Cell. Biol. 186, 201–209 CrossRef PubMed 32 Pitt, S.J., Funnell, T.M., Sitsapesan, M., Venturi, E., Rietdorf, K., Ruas, M., Ganesan, A., Gosain, R., Churchill, G.C., Zhu, M.X. et al. (2010) TPC2 is a novel NAADP-sensitive Ca2 + release channel, operating as a dual sensor of luminal pH and Ca2 + . J. Biol. Chem. 285, 35039–35046 CrossRef PubMed 33 Yamaguchi, S., Jha, A., Li, Q., Soyombo, A.A., Dickinson, G.D., Churamani, D., Brailoiu, E., Patel, S. and Muallem, S. (2010) Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels. J. Biol. Chem. 286, 22934–22942 CrossRef
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34 Grimm, C., Jors, S., Saldanha, S.A., Obukhov, A.G., Pan, B., Oshima, K., Cuajungco, M.P., Chase, P., Hodder, P. and Heller, S. (2010) Small molecule activators of TRPML3. Chem. Biol. 17, 135–148 CrossRef PubMed 35 Feng, X., Xiong, J., Lu, Y., Xia, X. and Zhu, M.X. (2014) Differential mechanisms of action of the mucolipin synthetic agonist, ML-SA1, on insect TRPML and mammalian TRPML1. Cell Calcium 56, 446–456 CrossRef 36 Xu, H., Delling, M., Li, L., Dong, X. and Clapham, D.E. (2007) Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice. Proc. Natl. Acad. Sci. U. S. A. 104, 18321–18326 CrossRef PubMed 37 Chen, C.C., Keller, M., Hess, M., Schiffmann, R., Urban, N., Wolfgardt, A., Schaefer, M., Bracher, F., Biel, M., Wahl-Schott, C. and Grimm, C. (2014) A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV. Nat. Commun. 5, 4681 PubMed 38 Lloyd-Evans, E., Morgan, A.J., He, X., Smith, D.A., Elliot-Smith, E., Sillence, D.J., Churchill, G.C., Schuchman, E.H., Galione, A. and Platt, F.M. (2008) Niemann–Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nat. Med. 14, 1247–1255 CrossRef PubMed 39 Lee, H., Lee, J.K., Min, W.K., Bae, J.H., He, X., Schuchman, E.H., Bae, J.S. and Jin, H.K. (2010) Bone marrow-derived mesenchymal stem cells prevent the loss of Niemann–Pick type C mouse Purkinje neurons by correcting sphingolipid metabolism and increasing sphingosine-1-phosphate. Stem Cells 28, 821–831 CrossRef PubMed 40 Xu, M., Liu, K., Swaroop, M., Porter, F.D., Sidhu, R., Firnkes, S., Ory, D.S., Marugan, J.J., Xiao, J., Southall, N. et al. (2012) delta-Tocopherol reduces lipid accumulation in Niemann–Pick type C1 and Wolman cholesterol storage disorders. J. Biol. Chem. 287, 39349–39360 CrossRef PubMed 41 Visentin, S., De Nuccio, C., Bernardo, A., Pepponi, R., Ferrante, A., Minghetti, L. and Popoli, P. (2013) The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann–Pick type C patients. J. Neurosci. 33, 15388–15393 CrossRef PubMed 42 Bae, M., Patel, N., Xu, H., Lee, M., Tominaga-Yamanaka, K., Nath, A., Geiger, J., Gorospe, M., Mattson, M.P. and Haughey, N.J. (2014) Activation of TRPML1 clears intraneuronal Abeta in preclinical models of HIV infection. J. Neurosci. 34, 11485–11503 CrossRef PubMed 43 He, X., Huang, Y., Li, B., Gong, C.X. and Schuchman, E.H. (2010) Deregulation of sphingolipid metabolism in Alzheimer’s disease. Neurobiol. Aging 31, 398–408 CrossRef PubMed Received 18 December 2014 doi:10.1042/BST20140311
