Transient Receptor Potential Mucolipin Research Articles

Emerging Role of TRPML1 Mucolipin Endolysosomal Channel in Cancer

The transient receptor potential mucolipin 1 (TRPML1) is an endolysosomal channel belonging to the TRP family. Clinically, mutations of TRPML1 have been responsible for a severe lysosomal storage disorder called mucolipidosis type IV.

Pathophysiological Role of Transient Receptor Potential Mucolipin Channel 1 in Calcium-Mediated Stress-Induced Neurodegenerative Diseases.

Mucolipins (TRPML) are endosome/lysosome Ca2+ permeable channels belonging to the family of transient receptor potential channels. In mammals, there are three TRPML proteins, TRPML1, 2, and 3, encoded by MCOLN1-3 genes. Among these channels, TRPML1 is a reactive oxygen species sensor localized on the lysosomal membrane that is able to control intracellular oxidative stress due to the activation of the autophagic process. Moreover, genetic or pharmacological inhibition of the TRPML1 channel stimulates oxidative stress signaling pathways. Experimental data suggest that elevated levels of reactive species play a role in several neurological disorders. There is a need to gain better understanding of the molecular mechanisms behind these neurodegenerative diseases, considering that the main sources of free radicals are mitochondria, that mitochondria/endoplasmic reticulum and lysosomes are coupled, and that growing evidence links neurodegenerative diseases to the gain or loss of function of proteins related to lysosome homeostasis. This review examines the significant roles played by the TRPML1 channel in the alterations of calcium signaling responsible for stress-mediated neurodegenerative disorders and its potential as a new therapeutic target for ameliorating neurodegeneration in our ever-aging population.

Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models.

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca2+ channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mdx mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca2+ channels may represent a promising approach to treat DMD and related muscle diseases.

Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

A lysosomal K+ channel regulates large particle phagocytosis by facilitating lysosome Ca2+ release

Macrophages are highly specialized in removing large particles including dead cells and cellular debris. When stimulated, delivery of the intracellular lysosomal membranes is required for the formation of plasmalemmal pseudopods and phagosomes. As a key lysosomal Ca2+ channel, Transient Receptor Potential Mucolipin-1 (TRPML1) regulates lysosomal exocytosis and subsequent phagosome biogenesis, thereby promoting phagocytosis of large extracellular particles. Recently, we have suggested that TRPML1-mediated lysosomal exocytosis is essentially dependent on lysosomal big conductance Ca2+-activated potassium (BK) channel. Therefore, we predict that lysosomal BK channels regulate large particle phagocytosis. In this study, by using RAW264.7 macrophage cell line and bone marrow-derived macrophages, we show that although BK is dispensable for small particle uptake, loss of BK significantly inhibits the ingestion of large particles whereas activating BK increases the uptake of large particles. BK facilitating effect on large particle ingestion is inhibited by either blocking TRPML1 or suppressing lysosomal exocytosis. Additionally, the increased uptake of large particles by activating TRPML1 is eliminated by inhibiting BK. These data suggest that BK and TRPML1 are functionally coupled to regulate large particle phagocytosis through modulating lysosomal exocytosis.

BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation

HIV-1 Tat is essential for HIV-1 replication and plays an important role in latent HIV-1 infection, HIV-1 associated neurological complication, and other HIV-1 comorbidities. Secreted from HIV-1 infected or transfected cells, Tat can be up-taken into cells by receptor-mediated endocytosis and internalized into endolysosomes. To reach nucleus where it can facilitate HIV-1 viral replication, exogenous Tat has to escape the degradation by endolysosomes. Because of findings that endolysosome de-acidification with, for example, the weak-base anti-malarial drug chloroquine prevents exogenous Tat degradation and enhances the amount of Tat available to activate HIV-1 LTR, we hypothesize that acidifying endolysosomes may enhance Tat degradation in endolysosomes and restrict LTR transactivation. Here, we determined the involvement of endolysosome-resident transient receptor potential mucolipin 1 channel (TRPML1) and the big conductance Ca2+-activated potassium (BK) channel in regulating endolysosome pH, as well as Tat-mediated HIV-1 LTR transactivation in U87MG cells stably integrated with HIV-1 LTR luciferase reporter. Activating TRPML1 channels with ML-SA1 acidified endolysosomes and restricted Tat-mediated HIV-1 LTR transactivation. These effects of ML-SA1 appeared to be mediated through activation of BK channels, because the effects of ML-SA1 on Tat-mediated HIV-1 LTR transactivation were blocked using pharmacological inhibitors or shRNA knock-down of BK channels. On the other hand, activating TRPML1 and BK channels enhanced cellular degradation of exogenous Tat. These results suggest that acidifying endolysosomes by activating TRPML1 or BK channels may provide therapeutic benefit against latent HIV-1 infection, HIV-1 associated neurocognitive disorders, and other HIV-1 comorbidities.

Control of lysosomal TRPML1 channel activity and exosome release by acid ceramidase in mouse podocytes.

The 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 whether the TRPML1 channel in podocytes mediates lysosome trafficking, which is essential for multivesicular body (MVB) degradation by lysosomes. We first demonstrated the abundant expression of TRPML1 channel in podocytes. By GCaMP3 Ca2+ imaging, we characterized the lysosomal specificity of TRPML1 channel-mediated Ca2+ release in podocytes. Given the important role of acid ceramidase (AC) in lysosome function and podocyte injury, we tested whether AC regulates this TRPML1 channel-mediated Ca2+ release and consequent lysosome-dependent MVB degradation in podocytes. Pharmacologically, it was found that TRPML1 channel activity was remarkably attenuated by the AC inhibitor carmofur. Sphingosine, as an AC product, was demonstrated to induce TRPML1-mediated Ca2+ release, which was inhibited by a TRPML1 blocker, verapamil. Using a Port-a-Patch planar patch-clamp system, we found that AC-associated sphingolipids, sphingomyelin, ceramide, and sphingosine had different effects on TRPML1 channel activity in podocytes. Functionally, the inhibition of AC or blockade of TRPML1 channels was found to suppress the interaction of lysosomes and MVBs, leading to increased exosome release from podocytes. These results suggest that AC is critical for TRPML1 channel-mediated Ca2+ release, which controls lysosome-MVB interaction and exosome release in podocytes.

Structure of the Human TRPML2 Ion Channel Extracytosolic/Lumenal Domain

TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between transmembrane helices S1 and S2. We present crystal structures of the tetrameric human TRPML2 ELD at pH 6.5 (2.0Å) and 4.5 (2.95Å), corresponding to the pH values in recycling endosomes and lysosomes. Isothermal titration calorimetry shows Ca2+ binding to the highly acidic central pre-pore loop which is abrogated at low pH, in line with a pH-dependent channel regulation model. Small angle X-ray scattering confirms the ELD dimensions in solution. Changes in pH or Ca2+ concentration do not affect the protein's secondary structure, but can influence ELD oligomer integrity according to native mass spectrometry. Our data thus complete the set of high-resolution views of human TRPML channel ELDs and reveal some structural responses to the conditions the TRPML2 ELD encounters as the channel traffics through the endolysosomal system.

Increased Lysosomal Exocytosis Induced by Lysosomal Ca2+ Channel Agonists Protects Human Dopaminergic Neurons from α-Synuclein Toxicity.

The accumulation of misfolded proteins is a common pathological feature of many neurodegenerative disorders, including synucleinopathies such as Parkinson's disease (PD), which is characterized by the presence of α-synuclein (α-syn)-containing Lewy bodies. However, although recent studies have investigated α-syn accumulation and propagation in neurons, the molecular mechanisms underlying α-syn transmission have been largely unexplored. Here, we examined a monogenic form of synucleinopathy caused by loss-of-function mutations in lysosomal ATP13A2/PARK9. These studies revealed that lysosomal exocytosis regulates intracellular levels of α-syn in human neurons. Loss of PARK9 function in patient-derived dopaminergic neurons disrupted lysosomal Ca2+ homeostasis, reduced lysosomal Ca2+ storage, increased cytosolic Ca2+, and impaired lysosomal exocytosis. Importantly, this dysfunction in lysosomal exocytosis impaired α-syn secretion from both axons and soma, promoting α-syn accumulation. However, activation of the lysosomal Ca2+ channel transient receptor potential mucolipin 1 (TRPML1) was sufficient to upregulate lysosomal exocytosis, rescue defective α-syn secretion, and prevent α-syn accumulation. Together, these results suggest that intracellular α-syn levels are regulated by lysosomal exocytosis in human dopaminergic neurons and may represent a potential therapeutic target for PD and other synucleinopathies.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is the second most common neurodegenerative disease linked to the accumulation of α-synuclein (α-syn) in patient neurons. However, it is unclear what the mechanism might be. Here, we demonstrate a novel role for lysosomal exocytosis in clearing intracellular α-syn and show that impairment of this pathway by mutations in the PD-linked gene ATP13A2/PARK9 contributes to α-syn accumulation in human dopaminergic neurons. Importantly, upregulating lysosomal exocytosis by increasing lysosomal Ca2+ levels was sufficient to rescue defective α-syn secretion and accumulation in patient neurons. These studies identify lysosomal exocytosis as a potential therapeutic target in diseases characterized by the accumulation of α-syn, including PD.

Cathepsin B Affects the Activation of Nucleotide-binding Domain and Leucine-rich-repeat-containing Family and Pyrin Domain-containing 3 Inflammasome via Transient Receptor Potential Mucolipin-1

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(H2O2),calcium-sensitive receptor agonist gadolinium trichloride(GdCl3),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of H2O2.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without H2O2.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with H2O2,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl3(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the H2O2 group and H2O2+GdCl3 group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of H2O2 concentration were similar.H2O2-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of H2O2.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the H2O2 +siRNA treatment group and the H2O2 treatment group.<b>Conclusion</b> CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.

Transient Receptor Potential Mucolipin-1 Channels in Glioblastoma: Role in Patient's Survival.

A link between mucolipin channels and tumors has been recently suggested. Herein, we aim to investigate the transient receptor potential mucolipin (TRPML)-1 relevance in glioblastoma. The expression of this channel was evaluated via qRT-PCR and immunohistochemistry in biopsies from 66 glioblastoma patients and two human glioblastoma cell lines and compared to normal human brain, astrocytes, and epileptic tissues. The subcellular distribution of TRPML-1 was examined via confocal microscopy in the glioma cell lines. Then, to assess the role of TRPML-1, cell viability assays have been conducted in T98 and U251 cell lines treated with the specific TRPML-1 agonist, MK6-83. We found that MK6-83 reduced cell viability and induced caspase-3-dependent apoptosis. Indeed, the TRPML-1 silencing or the blockage of TRPML-1 dependent [Ca2+]i release abrogated these effects. In addition, exposure of glioma cells to the reactive oxygen species (ROS) inducer, carbonyl cyanide m-chlorophenylhydrazone (CCCP), stimulated a TRPML-1-dependent autophagic cell death, as demonstrated by the ability of the autophagic inhibitor bafilomycin A, the TRPML-1 inhibitor sphingomyelin, and the TRPML-1 silencing to completely inhibit the CCCP-mediated effects. To test a possible correlation with patient’s survival, Kaplan–Meier, univariate, and multivariate analysis have been performed. Data showed that the loss/reduction of TRPML-1 mRNA expression strongly correlates with short survival in glioblastoma (GBM) patients, suggesting that the reduction of TRPML-1 expression represents a negative prognostic factor in GBM patients.

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.

Retraction: Curcumin Exerts Effects on the Pathophysiology of Alzheimer's Disease by Regulating PI(3,5)P2 and Transient Receptor Potential Mucolipin-1 Expression.

[This retracts the article DOI: 10.3389/fneur.2017.00531.].

Lipid⁻Protein Interactions in Niemann⁻Pick Type C Disease: Insights from Molecular Modeling.

The accumulation of lipids in the late endosomes and lysosomes of Niemann–Pick type C disease (NPCD) cells is a consequence of the dysfunction of one protein (usually NPC1) but induces dysfunction in many proteins. We used molecular docking to propose (a) that NPC1 exports not just cholesterol, but also sphingosine, (b) that the cholesterol sensitivity of big potassium channel (BK) can be traced to a previously unappreciated site on the channel’s voltage sensor, (c) that transient receptor potential mucolipin 1 (TRPML1) inhibition by sphingomyelin is likely an indirect effect, and (d) that phosphoinositides are responsible for both the mislocalization of annexin A2 (AnxA2) and a soluble NSF (N-ethylmaleimide Sensitive Fusion) protein attachment receptor (SNARE) recycling defect. These results are set in the context of existing knowledge of NPCD to sketch an account of the endolysosomal pathology key to this disease.

Defective autophagy and Alzheimer's disease: is calcium the key?

Defective autophagy and Alzheimer's disease: is calcium the key?

Endolysosomal Ca2+ Signalling and Cancer Hallmarks: Two-Pore Channels on the Move, TRPML1 Lags Behind!

The acidic vesicles of the endolysosomal (EL) system are emerging as an intracellular Ca2+ store implicated in the regulation of multiple cellular functions. The EL Ca2+ store releases Ca2+ through a variety of Ca2+-permeable channels, including Transient Receptor Potential (TRP) Mucolipin 1-3 (TRPML1-3) and two-pore channels 1-2 (TPC1-2), whereas EL Ca2+ refilling is sustained by the proton gradient across the EL membrane and/or by the endoplasmic reticulum (ER). EL Ca2+ signals may be either spatially restricted to control vesicle trafficking, autophagy and membrane repair or may be amplified into a global Ca2+ signal through the Ca2+-dependent recruitment of ER-embedded channels. Emerging evidence suggested that nicotinic acid adenine dinucleotide phosphate (NAADP)-gated TPCs sustain multiple cancer hallmarks, such as migration, invasiveness and angiogenesis. Herein, we first survey the EL Ca2+ refilling and release mechanisms and then focus on the oncogenic role of EL Ca2+ signaling. While the evidence in favor of TRPML1 involvement in neoplastic transformation is yet to be clearly provided, TPCs are emerging as an alternative target for anticancer therapies.

Transient Receptor Potential Ion Channel-Dependent Toxicity of Silica Nanoparticles and Poly(amido amine) Dendrimers.

Fundamental to the design and development of nanoparticles for applications in nanomedicine is a detailed understanding of their biologic fate and potential toxic effects. Transient receptor potential (TRP) ion channels are a large superfamily of cation channels with varied physiologic functions. This superfamily is classified into six related subfamilies: TRP canonical, TRP vanilloid (TRPV), TRP melastatin (TRPM), TRP ankyrin (TRPA), TRP polycystin, and TRP mucolipin. TRPA1, TRPM2, and TRPM8 are nonselective Ca2+-permeable cation channels which regulate calcium pathways under oxidative stress, whereas TRPV4 can be activated by oxidative, osmotic, and thermal stress as well as different fatty acid metabolites. Using a series of well characterized silica nanoparticles with variations in size (approximately 50-350 nm in diameter) and porosity, as well as cationic and anionic poly(amido amine) (PAMAM) dendrimers of similar size, we examined the toxicity of these nanoparticles to human embryonic kidney-293 cells overexpressing different TRP channels. The data show that the toxicity of mesoporous silica nanoparticles was influenced by expression of the TRPA1 and TRPM2 channels, whereas the toxicity of smaller nonporous silica nanoparticles was only affected by TRPM8. Additionally, TRPA1 and TRPM2 played a role in the cytotoxicity of cationic dendrimers, but not anionic dendrimers. TRPV4 did not seem to play a significant role in silica nanoparticle or PAMAM toxicity.

Structural basis for PtdInsP2-mediated human TRPML1 regulation

Transient receptor potential mucolipin 1 (TRPML1), a lysosomal channel, maintains the low pH and calcium levels for lysosomal function. Several small molecules modulate TRPML1 activity. ML-SA1, a synthetic agonist, binds to the pore region and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a natural lipid, stimulates channel activity to a lesser extent than ML-SA1; moreover, PtdIns(4,5)P2, another natural lipid, prevents TRPML1-mediated calcium release. Notably, PtdIns(3,5)P2 and ML-SA1 cooperate further increasing calcium efflux. Here we report the structures of human TRPML1 at pH 5.0 with PtdIns(3,5)P2, PtdIns(4,5)P2, or ML-SA1 and PtdIns(3,5)P2, revealing a unique lipid-binding site. PtdIns(3,5)P2 and PtdIns(4,5)P2 bind to the extended helices of S1, S2, and S3. The phosphate group of PtdIns(3,5)P2 induces Y355 to form a π-cation interaction with R403, moving the S4–S5 linker, thus allosterically activating the channel. Our structures and electrophysiological characterizations reveal an allosteric site and provide molecular insight into how lipids regulate TRP channels.

Identification of a single aspartate residue critical for both fast and slow calcium-dependent inactivation of the human TRPML1 channel

Transient receptor potential mucolipin subfamily 1 (TRPML1) is a nonselective cation channel mainly located in late endosomes and lysosomes. Mutations of the gene encoding human TRPML1 can cause severe lysosomal diseases. The activity of TRPML1 is regulated by both Ca2+ and H+, which are important for its critical physiological functions in membrane trafficking, exocytosis, autophagy, and intracellular signal transduction. However, the molecular mechanism of its dual regulation by Ca2+ and H+ remains elusive. Here, using a mutant screening method in combination with a whole-cell patch clamp technique, we identified a key TRPML1 residue, Asp-472, responsible for both fast calcium-dependent inactivation (FCDI) and slow calcium-dependent inactivation (SCDI) as well as H+ regulation. We also found that, in acidic pH, H+ can significantly delay FCDI and abolish SCDI and thereby presumably facilitate the ion conductance of the human TRPML1 channel. In summary, we have identified a key residue critical for Ca2+-induced inhibition of TRPML1 channel currents and uncovered pH-dependent regulation of this channel, providing vital information regarding the detailed mechanism of action of human TRPML1.

MOLEonline: a web-based tool for analyzing channels, tunnels and pores (2018 update).

MOLEonline is an interactive, web-based application for the detection and characterization of channels (pores and tunnels) within biomacromolecular structures. The updated version of MOLEonline overcomes limitations of the previous version by incorporating the recently developed LiteMol Viewer visualization engine and providing a simple, fully interactive user experience. The application enables two modes of calculation: one is dedicated to the analysis of channels while the other was specifically designed for transmembrane pores. As the application can use both PDB and mmCIF formats, it can be leveraged to analyze a wide spectrum of biomacromolecular structures, e.g. stemming from NMR, X-ray and cryo-EM techniques. The tool is interconnected with other bioinformatics tools (e.g., PDBe, CSA, ChannelsDB, OPM, UniProt) to help both setup and the analysis of acquired results. MOLEonline provides unprecedented analytics for the detection and structural characterization of channels, as well as information about their numerous physicochemical features. Here we present the application of MOLEonline for structural analyses of α-hemolysin and transient receptor potential mucolipin 1 (TRMP1) pores. The MOLEonline application is freely available via the Internet at https://mole.upol.cz.