Xian Ping Dong

PI(3,5)P 2 controls membrane trafficking by direct activation of mucolipin Ca 2+ release channels in the endolysosome

Membrane fusion and fission events in intracellular trafficking are controlled by both intraluminal Ca(2+) release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca(2+) release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P(2), an endolysosome-specific PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specificity and potency. Both PI(3,5)P(2)-deficient cells and cells that lack TRPML1 exhibited enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathway. We find that the enlarged vacuole phenotype observed in PI(3,5)P(2)-deficient mouse fibroblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P(2)-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca(2+) release from the vacuole. In this study, we show that this release requires both PI(3,5)P(2) production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane trafficking by transducing information regarding PI(3,5)P(2) levels into changes in juxtaorganellar Ca(2+), thereby triggering membrane fusion/fission events.

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe3+-bound transferrin receptors, or after lysosomal degradation of ferritin–iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe2+ transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe2+ transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe2+ permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe2+ at varying degrees, which correlate well with the disease severity. A comparison of TRPML1-/- ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe2+ levels, an increase in intralysosomal Fe2+ levels and an accumulation of lipofuscin-like molecules in TRPML1-/- cells. We propose that TRPML1 mediates a mechanism by which Fe2+ is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.

TPC Proteins Are Phosphoinositide- Activated Sodium-Selective Ion Channels in Endosomes and Lysosomes

Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.

Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release

Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1s expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

TRP Channel regulates EGFR signaling in Hair Morphogenesis and Skin Barrier Formation

A plethora of growth factors regulate keratinocyte proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that keratinocyte TRPV3, a member of the transient receptor potential (TRP) family of Ca(2+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(2+)-dependent crosslinking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.

TRP channels of intracellular membranes.

J. Neurochem. (2010) 113, 313–328.

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 (TRPML1V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1V432P-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.

Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

P2X4 and calmodulin form a signaling complex in late endosomes and lysosomes that promotes fusion and vacuolation in a Ca2+-dependent fashion.

P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH

Background: Ca2+-permeable P2X4 channels are expressed in lysosomes. Results: Lysosomal P2X4 channels are activated by ATP from the luminal side in a pH-dependent manner. Conclusion: P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Significance: Expanding the research of lysosomal Ca2+ signaling by adding another Ca2+ permeable channel on the lysosomal membranes. P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. The properties of these channels have been extensively studied on the plasma membrane. However, studies in amoeba suggest that P2X receptors are also present intracellularly and involved in vesicle fusion with the plasma membrane. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are activated and regulated. In this paper, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of COS1 and HEK293 cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrated that lysosomal P2X4 formed channels activated by ATP from the luminal side in a pH-dependent manner. While the acidic pH at the luminal side inhibited P2X4 activity, increasing the luminal pH in the presence of ATP caused P2X4 activation. We further showed that, as for the plasma membrane P2X4, the lysosomal P2X4 was potentiated by ivermectin but insensitive to suramin and PPADS, and it permeated the large cation N-methyl-d-glucamine upon activation. Our data suggest that P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Together with the reported fusion effect of intracellular P2X in lower organisms, we speculate that the lysosome-localized P2X4 may play specific roles in membrane trafficking of acidic organelles in mammalian cells.

SLC17A9 protein functions as a lysosomal ATP transporter and regulates cell viability.

Background: Lysosomes contain abundant ATP. Results: SLC17A9 transports ATP into lysosomes. SLC17A9-deficient cells exhibit lysosome dysfunction and undergo subsequent cell death. Conclusion: SLC17A9 functions as a lysosomal ATP transporter and maintains cell viability. Significance: This work identifies a molecular mechanism underlying lysosomal ATP accumulation and suggests that ATP functions not only as energy currency but plays a key role in lysosome function. Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability.