Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1

Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.     

Chaperone‐mediated autophagy is defective in mucolipidosis type IV

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin‐1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two‐hybrid and co‐immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone‐mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex‐ and age‐matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal‐associated membrane protein type 2A (LAMP‐2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly‐Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV. J. Cell. Physiol. 219: 344–353, 2009. © 2008 Wiley‐Liss, Inc.     

TRPML: transporters of metals in lysosomes essential for cell survival?

Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca²⁺-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe²⁺, Ca²⁺ and Zn²⁺ transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.     

Mucolipins: Intracellular TRPML1-3 channels

The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal “recycling center” for biological “garbage”, are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca²⁺/Fe²⁺) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration.     

Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane

Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca²⁺ and Fe²⁺ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P₂-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca²⁺, Mn²⁺, and Fe²⁺, but not Fe³⁺. The TRPML currents were inhibited by trivalent cations Fe³⁺, La³⁺, and Gd³⁺. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.     

Roles of CUP-5, the <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> orthologue of human TRPML1, in lysosome and gut granule biogenesis

Background  

CUP-5 is a Transient Receptor Potential protein in C. elegans that is the orthologue of mammalian TRPML1. Loss of TRPML1 results in the lysosomal storage disorder Mucolipidosis type IV. Loss of CUP-5 results in embryonic lethality and the accumulation of enlarged yolk granules in developing intestinal cells. The embryonic lethality of cup-5 mutants is rescued by mutations in mrp-4, which is required for gut granule differentiation. Gut granules are intestine-specific lysosome-related organelles that accumulate birefringent material. This link between CUP-5 and gut granules led us to determine the roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells.     

Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration, and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases. Here we report that an acute siRNA-mediated loss of TRPML1 specifically causes a leak of lysosomal protease cathepsin B (CatB) into the cytoplasm. CatB leak is associated with apoptosis, which can be prevented by CatB inhibition. Inhibition of the proapoptotic protein Bax prevents TRPML1 KD-mediated apoptosis but does not prevent cytosolic release of CatB. This is the first evidence of a mechanistic link between acute TRPML1 loss and cell death.     

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.     

Lysosomal trafficking functions of mucolipin-1 in murine macrophages

Background  

Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes.     

Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue

Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP-5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients mat not be sufficient to alleviate the symptoms due to tissue degeneration.     

Functional multimerization of mucolipin channel proteins

MCOLN1 encodes mucolipin‐1 (TRPML1), a member of the transient receptor potential TRPML subfamily of channel proteins. Mutations in MCOLN1 cause mucolipidosis‐type IV (MLIV), a lysosomal storage disorder characterized by severe neurologic, ophthalmologic, and gastrointestinal abnormalities. Along with TRPML1, there are two other TRPML family members, mucolipin‐2 (TRPML2) and mucolipin‐3 (TRPML3). In this study, we used immunocytochemical analysis to determine that TRPML1, TRPML2, and TRPML3 co‐localize in cells. The multimerization of TRPML proteins was confirmed by co‐immunoprecipitation and Western blot analysis, which demonstrated that TRPML1 homo‐multimerizes as well as hetero‐multimerizes with TRPML2 and TRPML3. MLIV‐causing mutants of TRPML1 also interacted with wild‐type TRPML1. Lipid bilayer re‐constitution of in vitro translated TRPML2 and TRPML3 confirmed their cation channel properties with lower single channel conductance and higher partial permeability to anions as compared to TRPML1. We further analyzed the electrophysiological properties of single channel TRPML hetero‐multimers, which displayed functional differences when compared to individual TRPMLs. Our data shows for the first time that TRPMLs form distinct functional channel complexes. Homo‐ and hetero‐multimerization of TRPMLs may modulate channel function and biophysical properties, thereby increasing TRPML functional diversity. J. Cell. Physiol. 222: 328–335, 2010. © 2009 Wiley‐Liss, Inc.     

Constitutive Activity of the Human TRPML2 Channel Induces Cell Degeneration

The mucolipin (TRPML) ion channel proteins represent a distinct subfamily of channel proteins within the transient receptor potential (TRP) superfamily of cation channels. Mucolipin 1, 2, and 3 (TRPML1, -2, and -3, respectively) are channel proteins that share high sequence homology with each other and homology in the transmembrane domain with other TRPs. Mutations in the TRPML1 protein are implicated in mucolipidosis type IV, whereas mutations in TRPML3 are found in the varitint-waddler mouse. The properties of the wild type TRPML2 channel are not well known. Here we show functional expression of the wild type human TRPML2 channel (h-TRPML2). The channel is functional at the plasma membrane and characterized by a significant inward rectification similar to other constitutively active TRPML mutant isoforms. The h-TRPML2 channel displays nonselective cation permeability, which is Ca²⁺-permeable and inhibited by low extracytosolic pH but not Ca²⁺ regulated. In addition, constitutively active h-TRPML2 leads to cell death by causing Ca²⁺ overload. Furthermore, we demonstrate by functional mutation analysis that h-TRPML2 shares similar characteristics and structural similarities with other TRPML channels that regulate the channel in a similar manner. Hence, in addition to overall structure, all three TRPML channels also share common modes of regulation.     

Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis

The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1^V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1^V432P-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca²⁺-dependent, constitutive Ca²⁺ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca²⁺ release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca²⁺ and Fe²⁺/Mn²⁺ dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.     

Mucolipidosis type IV: The importance of functional lysosomes for efficient autophagy

Mucolipidosis IV (MLIV) is a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. In contrast with most lysosomal storage disorders, which are attributed to the absence of specific lysosomal hydrolases, accumulation of material in MLIV results from defects in membrane transport along the late endocytic pathway. Mutations in MCOLN1 are the cause of MLIV; however, how lack of MCOLN1 function ultimately leads to neurodegeneration remains largely unknown. We found that MCOLN1 is required for efficient fusion of both late endosomes and autophagosomes with lysosomes. Impaired autophagosome degradation results in accumulation of autophagosomes in MLIV fibroblasts. In addition, we found increased levels and aggregation of p62, suggesting that abnormal accumulation of ubiquitinated protein inclusions may contribute to the neurodegenerative phenotype observed in MLIV patients. These findings corroborate recent evidence indicating that defects in autophagy may be a common feature of many neurodegenerative disorders.

Addendum to: Vergarajauregui S, Connelly PS, Daniels MP, and Puertollano R. Autophagic dysfunction in mucolipidosis type IV patients. Hum Mol Genet 2008; DOI: 10.1093/hmg/ddn174.     

Cellular Zinc Levels Are Modulated by TRPML1–TMEM163 Interaction

Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)‐163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two‐hybrid, tissue expression pattern, co‐immunoprecipitation, mass spectrometry and confocal microscopy studies confirmed the physical association of TMEM163 with TRPML1. This interaction is disrupted when a part of TMEM163's N‐terminus was deleted. Further studies to define the relevance of their interaction revealed that the plasma membrane (PM) levels of TMEM163 significantly decrease when TRPML1 is co‐expressed in HEK‐293 cells, while it mostly localizes within the PM when co‐expressed with a mutant TRPML1 that distributes mostly in the PM. Meanwhile, co‐expression of TMEM163 does not alter TRPML1 channel activity, but its expression levels in MLIV patient fibroblasts are reduced, which correlate with marked accumulation of zinc in lysosomes when these cells are acutely exposed to exogenous zinc (100 μM). When TMEM163 is knocked down or when TMEM163 and TRPML1 are co‐knocked down in HEK‐293 cells treated overnight with 100 nm zinc, the cells have significantly higher intracellular zinc levels than untreated control. Overall, these findings suggest that TMEM163 and TRPML1 proteins play a critical role in cellular zinc homeostasis, and thus possibly explain a novel mechanism for the pathological overload of zinc in MLIV disease.      

DNA damage and cell cycle events implicate cerebellar dentate nucleus neurons as targets of Alzheimer's disease

Background

The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.

Results

We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP), which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b). Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression.

Conclusions

These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered early in disease. Genetic and functional studies have implicated both LRP1 and NYGGF4 in obesity and cardiovascular disease and the physical association of these proteins may reflect a common mechanism. This is particularly interesting in light of the dual role of ApoE in both cardiovascular risk and AD. The results support further studies on the functional relationship between NYGGF4 and LRP1.     

Unc5B Interacts with FLRT3 and Rnd1 to Modulate Cell Adhesion in Xenopus Embryos

The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development.     

The protein interaction networks of mucolipins and two-pore channels

BackgroundThe endolysosomal, non-selective cation channels, two-pore channels (TPCs) and mucolipins (TRPMLs), regulate intracellular membrane dynamics and autophagy. While partially compensatory for each other, isoform-specific intracellular distribution, cell-type expression patterns, and regulatory mechanisms suggest different channel isoforms confer distinct properties to the cell.Scope of reviewBriefly, established TPC/TRPML functions and interaction partners (‘interactomes’) are discussed. Novel TRPML3 interactors are shown, and a meta-analysis of experimentally obtained channel interactomes conducted. Accordingly, interactomes are compared and contrasted, and subsequently described in detail for TPC1, TPC2, TRPML1, and TRPML3.Major conclusionsTPC interactomes are well-defined, encompassing intracellular membrane organisation proteins. TRPML interactomes are varied, encompassing cardiac contractility- and chaperone-mediated autophagy proteins, alongside regulators of intercellular signalling.General significanceComprising recently proposed targets to treat cancers, infections, metabolic disease and neurodegeneration, the advancement of TPC/TRPML understanding is of considerable importance. This review proposes novel directions elucidating TPC/TRPML relevance in health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases

Disrupted cellular Ca²⁺ signaling is believed to play a role in a number of human diseases including lysosomal storage diseases (LSD). LSDs are a group of ∼50 diseases caused predominantly by mutations in lysosomal proteins that result in accumulation of macromolecules within the lysosome. We recently reported that Niemann-Pick type C (NPC) is the first human disease to be associated with defective lysosomal Ca²⁺ uptake and defective NAADP-mediated lysosomal Ca²⁺ release. These defects in NPC cells leads to the disruption in endocytosis and subsequent lipid storage that is a feature of this disease. In contrast, Chediak-Higashi Syndrome cells have been reported to have enhanced lysosomal Ca²⁺ uptake whilst the TRPML1 protein defective in mucolipidosis type IV is believed to function as a Ca²⁺ channel. In this review we provide a summary of the current knowledge on the role of lysosomal Ca²⁺ signaling in the pathogenesis of this group of diseases.     

Differential mechanisms of action of the mucolipin synthetic agonist,ML-SA1, on insect TRPML and mammalian TRPML1

Mucolipin synthetic agonist 1 (ML-SA1) was recently identified to activate mammalian TRPML channels and shown to alleviate lipid accumulation in lysosomes of cellular models of lysosome storage diseases, mucolipidosis type IV (MLIV) and Niemann–Pick's disease type C (NPC). Owning to its potential use in complimenting genetic studies in Drosophila melanogaster to elucidate the cellular and physiological functions of TRPML channels, we examined the effect of ML-SA1 on Drosophila TRPML expressed in HEK293 cells using whole-cell, inside-out, and whole-lysosome electrophysiological recordings. We previously showed that when expressed in HEK293 cells, Drosophila TRPML was localized and functional on both plasma membrane and endolysosome. We show here that in both inside-out patches excised from the plasma membrane and whole-lysosome recordings from enlarged endolysosome vacuoles, ML-SA1 failed to activate TRPML unless exogenous phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] was applied. At 1 μM ML-SA1, the sensitivity of TRPML to PI(3,5)P₂ increased approximately by 10-fold and at 10 μM ML-SA1, the deactivation of PI(3,5)P₂-evoked TRPML currents was markedly slowed. On the other hand, constitutive activation of TRPML by a mutation that mimics the varitint-waddler (Va) mutation of mouse TRPML3 rendered the insect channel sensitive to activation by ML-SA1 alone. Moreover, different from the insect TRPML, mouse TRPML1 was readily activated by ML-SA1 independent of PI(3,5)P₂. Thus, our data reveal that while ML-SA1 acts as a true agonist at mouse TRPML1, it behaves as an allosteric activator of the Drosophila TRPML, showing dependence on and the ability to stabilize open conformation of the insect channels.