TRPML1 Research Articles

Enhanced Exosome Release and Inhibited TRPML1 Channel Activity in Podocytes from Mice with Podocyte‐Restricted Deletion of Asah1 Gene

The ceramide hydrolysis by lysosomal acid ceramidase (AC) has been reported to regulate lysosome function, which determines the degradation of multivesicular bodies. In this regard, we found that urinary exosome excretion was remarkably increased in podocyte‐specific AC gene knockout (Asah1fl/fl/Podocre) mice. Given the importance of transient receptor potential mucolipin 1 (TRPML1) channel‐mediated Ca2+ release to lysosome function and its regulation by sphingolipids, the present study tested a hypothesis that podocyte‐specific Asah1 gene deletion inhibits TRPML1 channel activity, leading to lysosome dysfunction and exosome release from podocytes. It was found that exosomes released from Asah1fl/fl/Podocre podocytes were remarkably higher than WT/WT podocytes as detected by nanoparticle tracking analysis. Super‐resolution microscopy showed less fusion of multivesicular bodies and lysosomes in Asah1fl/fl/Podocre podocytes compared with WT/WT podocytes. Using GCaMP3 Ca2+ imaging, it was found that ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased Ca2+ release from lysosomes by 7.35 folds in WT/WT podocytes. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in WT/WT podocytes. In Asah1fl/fl/Podocre podocytes, however, ML‐SA1 was found to have no effects on TRPML1 channel‐mediated Ca2+ release, but sphingosine as an AC product of ceramide hydrolysis remarkably increased TRPML1 channel‐mediated Ca2+ release in both WT/WT podocytes (5.42 folds) and Asah1fl/fl/Podocre podocytes (4.52 folds). Using whole‐lysosome patch clamp recording, we detected ML‐SA1‐induced Ca2+ currents in a dose‐dependent manner through the membrane of lysosomes isolated from WT/WT podocytes. Compared to WT/WT podocytes, intact lysosomes isolated from Asah1fl/fl/Podocre podocytes had evidently blunted opening of TRPML1 channels in response to its agonist, ML‐SA1. AC associated sphingolipids, sphingomyelin, ceramide and sphingosine had different effects on TRPML1 channel activity in WT/WT podocytes, with inhibition by sphingomyelin, no effect from ceramide, but enhancement by sphingosine (2.25 folds). In Asah1fl/fl/Podocre podocytes, sphingosine remained to increase ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release (3.96 folds). These results suggest that the AC deficiency decreases TRPML1 channel activity due to reduction of sphingosine production, which may contribute to lysosome dysfunction and enhanced exosome release.Support or Funding InformationThis study was supported by NIH grants DK54927, DK120491, and HL057244.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial respiratory chain deficiency inhibits lysosomal hydrolysis

ABSTRACTMitochondria are key organelles for cellular metabolism, and regulate several processes including cell death and macroautophagy/autophagy. Here, we show that mitochondrial respiratory chain (RC) deficiency deactivates AMP-activated protein kinase (AMPK, a key regulator of energy homeostasis) signaling in tissue and in cultured cells. The deactivation of AMPK in RC-deficiency is due to increased expression of the AMPK-inhibiting protein FLCN (folliculin). AMPK is found to be necessary for basal lysosomal function, and AMPK deactivation in RC-deficiency inhibits lysosomal function by decreasing the activity of the lysosomal Ca2+ channel MCOLN1 (mucolipin 1). MCOLN1 is regulated by phosphoinositide kinase PIKFYVE and its product PtdIns(3,5)P2, which is also decreased in RC-deficiency. Notably, reactivation of AMPK, in a PIKFYVE-dependent manner, or of MCOLN1 in RC-deficient cells, restores lysosomal hydrolytic capacity. Building on these data and the literature, we propose that downregulation of the AMPK-PIKFYVE-PtdIns(3,5)P2-MCOLN1 pathway causes lysosomal Ca2+ accumulation and impaired lysosomal catabolism. Besides unveiling a novel role of AMPK in lysosomal function, this study points to the mechanism that links mitochondrial malfunction to impaired lysosomal catabolism, underscoring the importance of AMPK and the complexity of organelle cross-talk in the regulation of cellular homeostasis.Abbreviation: ΔΨm: mitochondrial transmembrane potential; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATP: adenosine triphosphate; ATP6V0A1: ATPase, H+ transporting, lysosomal, V0 subbunit A1; ATP6V1A: ATPase, H+ transporting, lysosomal, V0 subbunit A; BSA: bovine serum albumin; CCCP: carbonyl cyanide-m-chlorophenylhydrazone; CREB1: cAMP response element binding protein 1; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; EBSS: Earl’s balanced salt solution; ER: endoplasmic reticulum; FBS: fetal bovine serum; FCCP: carbonyl cyanide-p-trifluoromethoxyphenolhydrazone; GFP: green fluorescent protein; GPN: glycyl-L-phenylalanine 2-naphthylamide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryonic fibroblast; MITF: melanocyte inducing transcription factor; ML1N*2-GFP: probe used to detect PtdIns(3,5)P2 based on the transmembrane domain of MCOLN1; MTORC1: mechanistic target of rapamycin kinase complex 1; NDUFS4: NADH:ubiquinone oxidoreductase subunit S4; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; pcDNA: plasmid cytomegalovirus promoter DNA; PCR: polymerase chain reaction; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; P/S: penicillin-streptomycin; PVDF: polyvinylidene fluoride; qPCR: quantitative real time polymerase chain reaction; RFP: red fluorescent protein; RNA: ribonucleic acid; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TMRM: tetramethylrhodamine, methyl ester, perchlorate; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; v-ATPase: vacuolar-type H+-translocating ATPase; WT: wild-type

Dysregulation of Microglial Function Contributes to Neuronal Impairment in Mcoln1a-Deficient Zebrafish

SummaryType IV mucolipidosis (ML-IV) is a neurodegenerative lysosome storage disorder caused by mutations in the MCOLN1 gene. However, the cellular and molecular bases underlying the neuronal phenotypes of ML-IV disease remain elusive. Using a forward genetic screening, we identified a zebrafish mutant, biluo, that harbors a hypomorphic mutation in mcoln1a, one of the two zebrafish homologs of mammalian MCOLN1. The mcoln1a-deficient mutants display phenotypes partially recapitulating the key features of ML-IV disorder, including the accumulation of enlarged late endosomes in microglia and aberrant neuronal activities in both spontaneous and visual-evoking conditions in optic tectal neurons. We further show that the accumulation of enlarged late endosomes in microglia is caused by the impairment of late endosome and lysosome fusion and the aberrant neuronal activities can be partially rescued by the reconstitution of Mcoln1a function in microglia. Our findings suggest that dysregulation of microglial function may contribute to the development and progression of ML-IV disease.

Current concepts in the neuropathogenesis of mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive, lysosomal storage disorder causing progressively severe intellectual disability, motor and speech deficits, retinal degeneration often culminating in blindness, and systemic disease causing a shortened lifespan. MLIV results from mutations in the gene MCOLN1 encoding the transient receptor potential channel mucolipin-1. It is an ultra-rare disease and is currently known to affect just over 100 diagnosed individuals. The last decade has provided a wealth of research focused on understanding the role of the enigmatic mucolipin-1 protein in cell and brain function and how its absence causes disease. This review explores our current understanding of the mucolipin-1 protein in relation to neuropathogenesis in MLIV and describes recent findings implicating mucolipin-1's important role in mechanistic target of rapamycin and TFEB (transcription factor EB) signaling feedback loops as well as in the function of the greater endosomal/lysosomal system. In addition to addressing the vital role of mucolipin-1 in the brain, we also report new data on the question of whether haploinsufficiency as would be anticipated in MCOLN1 heterozygotes is associated with any evidence of neuron dysfunction or disease. Greater insights into the role of mucolipin-1 in the nervous system can be expected to shed light not only on MLIV disease but also on numerous processes governing normal brain function. This article is part of the Special Issue "Lysosomal Storage Disorders".

Mucolipidosis type IV in a child

Mucolipidosis type IV is a rare autosomal recessive lysosomal storage disorder with psychomotor developmental delay, visual impairment, and achlorhydria. A mutation in the MCOLN1 gene causes an alteration of the protein mucolipin-1 that results in the accumulation of lipids and proteins in cytoplasmic vacuoles derived from lysosomes. Visual impairment results mainly from corneal clouding and retinal degeneration. The involvement of the corneal epithelium has been proposed following clinical observation and confirmed by ultrastructural studies of the cornea. We present the case of a child of French Canadian origin affected by mucolipidosis type IV who showed abnormal optical coherence tomography imaging of the cornea, typical skin cell inclusions on electronic microscopy, and a novel pathogenic mutation.

Fingolimod phosphate inhibits astrocyte inflammatory activity in mucolipidosis IV.

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

The regulatory mechanism of mammalian TRPMLs revealed by cryo-EM.

Transient receptor potential mucolipin (TRPML) channels are the most recently identified subfamily of TRP channels and have seen a surge of new reports revealing both structural and functional insight. In 2017, several groups published multiple conformations of TRPML channels using cryo-EM. Similar to other TRP channels, the ML subfamily consists of six transmembrane helices (S1-S6), and a pore region including S5, S6, and two pore helices (PH1 and PH2). However, these reports also reveal distinct structural characteristics of the ML subfamily. Asp residues within the luminal pore may function to control calcium/pH regulation. A synthetic agonist, ML-SA1, can bind to the pore region of TRPMLs to force a direct dilation of the lower gate. Finally, biophysical and electrophysiological characterizations reveal another natural agonist binding site in the unique domain of TRPMLs, presumably regulating the conformation of the S4-S5 linker to open the channel. This work elucidates the molecular architecture and provides insights into how multiple ligands regulate TRPMLs.

Lysosomal Ca2+ Release via TRPML1 Channels Regulated by Acid Ceramidase and Associated Sphingolipids in Podocytes

Transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca2+ release that is involved in Ca2+‐dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested the hypothesis that acid ceramidase (AC) plays a pivotal role in regulation of TRPML1 channel activity in podocytes. To detect TRPML1 channel‐mediated Ca2+ release, cultured murine podocytes were transfected via nucleofection with GCaMP3‐ML1, a TRPML1‐targeted genetically encoded Ca2+ indicator. To characterize GCaMP3 expression in lysosomes, confocal microscopy confirmed the colocalization of GCaMP3‐ML1 and Lamp‐1 suggesting its successful transfection and localization in lysosomes. ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased GCaMP3 fluorescence intensity in podocytes. For dynamic recording of Ca2+ release, a high speed wavelength switching fluorescent microscopic imaging system was used to continuously monitor GCaMP3 fluorescence signal in living podocytes. It was found that in the presence of a lysosome‐disrupting cathepsin C substrate, GPN that depleted lysosomal Ca2+, GCaMP3‐detected Ca2+ release response to ML‐SA1 was substantially attenuated. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in podocytes. Sphingosine as the AC product of ceramide hydrolysis was found to increase GCaMP3 fluorescence by 3.95 folds in podocytes. Whole‐lysosome patch clamp recording using a Port‐A‐Patch system was performed to study the effects of various AC‐associated sphingolipids on TRPML1 channel activity. After characterization of integrity and purity of isolated lysosomes, we detected ML‐SA1‐induced Ca2+ currents through the isolated lysosome membrane in a dose‐dependent manner. Sphingomyelin, the substrate of ceramide, blocked Ca2+ release induced by ML‐SA1, ceramide had no effects on ML‐SA1‐induced Ca2+ currents, but sphingosine as a product of ceramide hydrolysis via AC remarkably increased ML‐SA1‐induced Ca2+ release by 2.35 folds. Furthermore, interactions of lysosome and autophagosome in podocytes indicated by colocalization of LC3B‐GFP and Lamp‐1‐RFP were inhibited by carmofur. These results suggest that normal function of lysosomal AC and associated production of sphingosine are important for TRPML1 channel activity and Ca2+‐dependent lysosome trafficking.Support or Funding InformationThis study was supported by NIH grants DK54927 to Pin‐Lan Li and HL89563 to Ningjun Li.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PAX5‐Induced Expression of Endogenous Mucolipin‐2 (MCOLN2) Gene in Human Glial and Neuronal Cell Lines: A Potential Gene Complementation Therapy Approach for Mucolipidosis IV

The loss of function in the Transient Receptor Potential Mucolipin‐1 (TRPML1) protein, encoded by the Mucolipin‐1 (MCOLN1) gene, is known to cause the neurodegenerative disease Mucolipidosis type IV (MLIV). Characteristics of MLIV are motor problems, cognitive dysfunction, achlorhydria, cataracts, and blindness caused by retinal cell death. The Ashkenazi Jew population is most afflicted by the disease, and MLIV has no known cure. The Mucolipin ion channels consist of TRPML1, ‐2, and ‐3 proteins, which are encoded by MCOLN1, ‐2, and ‐3 genes, respectively. They are known to serve as non‐selective cation channels located within endosomes and lysosomes and play a role in endosome‐lysosome fusion. Previous research indicates that TRPML1 and TRPML2 proteins share a high degree of homology, and we hypothesize that TRPML2, which has no known association to clinical disease, could substitute for the loss of functional TRPML1 in MLIV, resulting in a rescue of the disease phenotype. In order to study the potential therapeutic effects of TRPML2 in MLIV, we must first show that it is expressed in the brain, one of the tissues most affected by MLIV. We have previously discovered PAX5 as the transcriptional activator for MCOLN2 and have shown that MCOLN2 expression could be heterologously induced in both human neuroglioma (H4) cells and human embryonic kidney 293 (HEK‐293) cell lines. Previous reports demonstrated that MCOLN2 has tissue‐specific expression with little to no detectable transcripts in brain tissue. However, our preliminary research has detected endogenous levels of MCOLN2 transcripts in human H4 and neuroblastoma (SH‐SY5Y) cells. Moreover, to induce the expression of endogenous MCOLN2, we transduced SH‐SY5Y cells with adenovirus and lentivirus vectors containing the PAX5 cDNA. PAX5 serves as a B cell‐specific activator protein with tissue‐specific expression pattern as well. Standard reverse‐transcription polymerase chain reaction (RT‐PCR) and real‐time quantitative RT‐PCR both revealed MCOLN2 expression in SH‐SY5Y cells at 48 and 72 hours post‐transduction. We show that PAX5 transcriptionally activates and increases endogenous MCOLN2 expression in neuronal cells that are central to studying MLIV. Future studies include In‐Cell Western assay with an anti‐TRPML2 antibody and transcriptome analysis of PAX5‐treated cells to determine global gene expression. These results and future studies will further explore the therapeutic potential of gene complementation to rescue the cellular phenotype in MLIV disease.Support or Funding InformationThis work was partly funded by NIH R15 NS101594.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Gene Expression Analysis of Brain Tissue RNA From Mucolipidosis IV Knockout Mice

Mucolipidosis IV (MLIV) is an autosomal recessive disorder that is characterized by progressive neuronal and retinal degeneration. MLIV is caused by loss of function mutations in the human Mucolipin‐1 (MCOLN1) gene, whose protein product, Transient Receptor Potential Mucolipin‐1 (TRPML1), serves as a lysosomal non‐selective cation channel. We have previously reported abnormal zinc levels in MLIV patient fibroblasts, human MCOLN1‐knockdown cells, and Mcoln1‐knockout (KO) mouse brain tissues. Collectively, these observations suggest that the loss of cellular zinc homeostasis may play an underlying role in MLIV pathogenesis. To investigate whether these irregularities are reflected in the gene expression patterns of zinc buffering and transport proteins, we performed RNA sequencing (RNA‐seq) to analyze the brain transcriptome of three Mcoln1 KO mice and three wild‐type (WT) littermate controls. Gene ontology data revealed significant differential gene expression in the category of “Zinc Ion Transmembrane Transporter Activity.” Gene cluster analyses of zinc transport and zinc buffering genes revealed a distinct reduction in brain transcript levels of solute carrier 30a3 (Slc30a3; also known as the efflux zinc transporter, ZnT3) and transmembrane (Tmem)‐163 genes in KO mice, both of which facilitate zinc transport within neuronal synaptic vesicles. Comparison of RNA‐seq data between individual assembly and combined assembly of KO and WT sequencing reads showed distinct transcript‐level variability that could be explained by inter‐subject differences. Validation using real‐time quantitative polymerase chain reaction (qPCR) confirmed this effect. Nevertheless, both RNA‐seq and qPCR results consistently showed a decrease in ZnT3 transcript levels in KO mice compared to WT littermate control mice. These data imply that abnormal ZnT3 expression could be a mitigating factor for the observed zinc dyshomeostasis in MLIV cells. Overall, our findings suggest that the loss of human TRPML1 protein function perturbs the expression of specific genes necessary for intracellular zinc homeostasis. Future studies will investigate whether the reduction of ZnT3 transcript levels also reflect decreased protein levels in brain tissues of MLIV KO versus control mice.Support or Funding InformationThis work was funded by NIH R15 NS101594 and NIH R25 MD010397.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial TFEB (Transcription Factor EB) Positively Regulates Postischemic Angiogenesis.

Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-α signaling and upregulated autophagy. Through inactivation of AMPKα or inhibition of autophagy, we demonstrated that the AMPKα and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPKα activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism

ABSTRACT Macroautophagy/autophagy is an evolutionarily conserved pathway that is required for cellular homeostasis, growth and survival. The lysosome plays an essential role in autophagy regulation. For example, the activity of MTORC1, a master regulator of autophagy, is regulated by nutrients within the lysosome. Starvation inhibits MTORC1 causing autophagy induction. Given that MTORC1 is critical for protein synthesis and cellular homeostasis, a feedback regulatory mechanism must exist to restore MTORC1 during starvation. However, the molecular mechanism underlying this feedback regulation is unclear. In this study, we report that starvation activates the lysosomal Ca2+ release channel MCOLN1 (mucolipin 1) by relieving MTORC1's inhibition of the channel. Activated MCOLN1 in turn facilitates MTORC1 activity that requires CALM (calmodulin). Moreover, both MCOLN1 and CALM are necessary for MTORC1 reactivation during prolonged starvation. Our data suggest that lysosomal Ca2+ signaling is an essential component of the canonical MTORC1-dependent autophagy pathway and MCOLN1 provides a negative feedback regulation of MTORC1 to prevent excessive loss of MTORC1 function during starvation. The feedback regulation may be important for maintaining cellular homeostasis during starvation, as well as many other stressful or disease conditions.

Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3.

SummaryThe modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signaling. The mucolipin transient receptor potential (TRPML) channel family belongs to the TRP superfamily1,2 and is composed of three members, TRPML1-3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate organelle Ca2+ releases important for various physiological processes, including organelle trafficking and fusion3. Loss-of-function mutations in the TRPML1 gene cause the neurodegenerative lysosomal storage disorder mucolipidosis IV (ML-IV), and a gain-of-function mutation in TRPML3 (Ala419Pro) gives rise to the Varitint-Waddler (Va) mouse phenotype4–6. Notably, TRPMLs are activated by the low-abundance and LEL-enriched signaling lipid PI(3,5)P2, while other phosphoinositides such as PI(4,5)P2, enriched in plasma membranes, inhibit TRPMLs7,8. Conserved basic residues at the N-terminus of the channels are important for PI(3,5)P2 activation and PI(4,5)P2 inhibition8. However, due to a lack of structural information, the mechanism by which TRPML channels recognize PI(3,5)P2 and increase its Ca2+ conductance remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3, at an average resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain we term the “mucolipin domain” (MLD). Combined with functional studies, we suggest that the MLD is responsible for PI(3,5)P2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs.

Transient receptor potential mucolipin 1 (TRPML1) is an endo/lysosomal cation channel ubiquitously expressed in mammalian cells1,2 and its loss-of-function mutations are the direct cause of Type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease3-6. Here we present the single particle cryo-electron microscopy (cryo-EM) structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis, the TRPML1 structure reveals that phosphatidylinositol bisphosphate (PIP2) binds to the N-terminus of the channel – distal from the pore – and the helix-turn-helix extension between S2 and S3 likely couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion binding sites and the conserved acidic residues form the luminal Ca2+ blocking site that confers luminal pH and Ca2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing multiple luminal ion passages to the pore and also creating a negative electrostatic trap – preferably for divalent cations at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, providing structural implication for the S4-S5 linker-mediated PIP2 gating mechanism among TRPML channels7,8.

Human TRPML1 channel structures in open and closed conformations.

Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis.

Leukocyte TRP channel gene expressions in\xa0patients with non-valvular atrial fibrillation

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and is a major cause of morbidity and mortality. The upregulation of TRP channels is believed to mediate the progression of electrical remodelling and the arrhythmogenesis of the diseased heart. However, there is limited data about the contribution of the TRP channels to development of AF. The aim of this study was to investigate leukocyte TRP channels gene expressions in non-valvular atrial fibrillation (NVAF) patients. The study included 47 NVAF patients and 47 sex and age matched controls. mRNA was extracted from blood samples, and real-time polymerase chain reaction was performed for gene expressions by using a dynamic array system. Low levels of TRP channel expressions in the controls were markedly potentiated in NVAF group. We observed marked increases in MCOLN1 (TRPML1), MCOLN2 (TRPML2), MCOLN3 (TRPML3), TRPA1, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, TRPV6, and PKD2 (TRPP2) gene expressions in NVAF patients (P < 0.05). However, there was no change in PKD1 (TRPP1) gene expression. This is the first study to provide evidence that elevated gene expressions of TRP channels are associated with the pathogenesis of NVAF.

139 An investigation into the subcellular distribution of two-pore channels in cardiac ventricular myocytes in light of their differing contributions to beta-adrenergic signalling

IntroductionNicotinic acid adenine dinucleotide phosphate (NAADP) is a calcium-mobilising messenger that acts via two-pore calcium channels (TPCs). NAADP releases calcium from a lysosome-related acidic compartment distinct from the endo-sarcoplasmic reticulum...

The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin

Intracellular lysosomal membrane trafficking, including fusion and fission, is crucial for cellular homeostasis and normal cell function. Both fusion and fission of lysosomal membrane are accompanied by lysosomal Ca2+ release. We recently have demonstrated that the lysosomal Ca2+ release channel P2X4 regulates lysosome fusion through a calmodulin (CaM)-dependent mechanism. However, the molecular mechanism underlying lysosome fission remains uncertain. In this study, we report that enlarged lysosomes/vacuoles induced by either vacuolin-1 or P2X4 activation are suppressed by up-regulating the lysosomal Ca2+ release channel transient receptor potential mucolipin 1 (TRPML1) but not the lysosomal Na+ release channel two-pore channel 2 (TPC2). Activation of TRPML1 facilitated the recovery of enlarged lysosomes/vacuoles. Moreover, the effects of TRPML1 on lysosome/vacuole size regulation were eliminated by Ca2+ chelation, suggesting a requirement for TRPML1-mediated Ca2+ release. We further demonstrate that the prototypical Ca2+ sensor CaM is required for the regulation of lysosome/vacuole size by TRPML1, suggesting that TRPML1 may promote lysosome fission by activating CaM. Given that lysosome fission is implicated in both lysosome biogenesis and reformation, our findings suggest that TRPML1 may function as a key lysosomal Ca2+ channel controlling both lysosome biogenesis and reformation.

Inhibition of Transient Receptor Potential Channel Mucolipin-1 (TRPML1) by Lysosomal Adenosine Involved in Severe Combined Immunodeficiency Diseases

Impaired adenosine homeostasis has been associated with numerous human diseases. Lysosomes are referred to as the cellular recycling centers that generate adenosine by breaking down nucleic acids or ATP. Recent studies have suggested that lysosomal adenosine overload causes lysosome defects that phenocopy patients with mutations in transient receptor potential channel mucolipin-1 (TRPML1), a lysosomal Ca2+ channel, suggesting that lysosomal adenosine overload may impair TRPML1 and then lead to subsequent lysosomal dysfunction. In this study, we demonstrate that lysosomal adenosine is elevated by deleting adenosine deaminase (ADA), an enzyme responsible for adenosine degradation. We also show that lysosomal adenosine accumulation inhibits TRPML1, which is rescued by overexpressing ENT3, the adenosine transporter situated in the lysosome membrane. Moreover, ADA deficiency results in lysosome enlargement, alkalinization, and dysfunction. These are rescued by activating TRPML1. Importantly, ADA-deficient B-lymphocytes are more vulnerable to oxidative stress, and this was rescued by TRPML1 activation. Our data suggest that lysosomal adenosine accumulation impairs lysosome function by inhibiting TRPML1 and subsequently leads to cell death in B-lymphocytes. Activating TRPML1 could be a new therapeutic strategy for those diseases.

BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases.

Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs.