Summary Mammalian Two-Pore Channels (TPC1, 2; 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)P2, 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)P2 in regulating the fusogenic potential of intracellular organelles.

Membrane fusion and fi ssion events in intracellular traffi cking 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-specifi c PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specifi city and potency. Both PI(3,5)P 2 -defi cient cells and cells that lack TRPML1 exhibited enlarged endolysosomes / vacuoles and traffi cking defects in the late endocytic pathway. We fi nd that the enlarged vacuole phenotype observed in PI(3,5)P 2 -defi cient mouse fi broblasts 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 traffi cking by transducing information regarding PI(3,5)P 2 levels into changes in juxtaorganellar Ca 2 + , thereby triggering membrane fusion / fi ssion events.

Lysosomal lipid accumulation, defects in membrane trafficking, and altered Ca2+ homeostasis are common features in many lysosomal storage diseases. Mucolipin TRP channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ 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 NP cell lysosomes. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP type C (NPC) cells, increasing TRPML1’s expression/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 Ca2+-dependent lysosomal trafficking.

TRPML1 (mucolipin-1/MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein belonging to the mucolipin subfamily of Transient Receptor Potential (TRP) proteins 1–3. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4) 4, 5. ML4 patients exhibit motor impairment, mental retardation, retinal degeneration, and iron-deficiency anemia. Since aberrant iron metabolism may cause neural and retinal degeneration 6, 7, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes following iron uptake via endocytosis of Fe3+-bound transferrin receptors 6, or following lysosomal degradation of ferritin-Fe complexes and autophagic ingestion of iron-containing macromolecules 6, 8, is the major source of cellular iron. The Divalent Metal Transporter protein (DMT1) is the only endosomal Fe2+ transporter currently known and is highly expressed in erythroid precursors 6, 9, but genetic studies suggest the existence of a DMT1-independent endosomal/lysosomal Fe2+ transport protein 9. Here, by measuring radiolabeled iron uptake, monitoring the levels of cytosolic and intra-lysosomal iron and directly patch-clamping the late endosomal/lysosomal membrane, we show that TRPML1 functions as a Fe2+ permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair TRPML1’s ability to permeate Fe2+ at varying degrees, which correlate well with the disease severity. A comparison of TRPML1−/− ML4 and control skin fibroblasts showed a reduction of cytosolic Fe2+ levels, an increase of intra-lysosomal 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/lysosomes. Our results suggest that impaired iron transport may contribute to both hematological and degenerative symptoms of ML4 patients.

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Transient receptor potential (TRP) genes of the mucolipin subfamily (TRPML1-3 and MCOLN1-3) are presumed to encode ion channel proteins of intracellular endosomes and lysosomes. Mutations in human TRPML1 (mucolipin 1/MCOLN1) result in mucolipidosis type IV, a severe inherited neurodegenerative disease associated with defective lysosomal biogenesis and trafficking. A mutation in mouse TRPML3 (A419P; TRPML3 Va ) results in the varitint-waddler (Va) phenotype. Va mice are deaf, exhibit circling behavior due to vestibular defects, and have variegated/dilute coat color as a result of pigmentation defects. Prior electrophysiological studies of presumed TRPML plasma membrane channels are contradictory and inconsistent with known TRP channel properties. Here, we report that the Va mutation produces a gain-of-function that allows TRPML1 and TRPML3 to be measured and identified as inwardly rectifying, proton-impermeant, Ca 2؉ -permeant cation channels. TRPML3 is highly expressed in normal melanocytes. Melanocyte markers are lost in the Va mouse, suggesting that their variegated and hypopigmented fur is caused by severe alteration of melanocyte function or cell death. TRPML3 Va expression in melanocyte cell lines results in high resting Ca 2؉ levels, rounded, poorly adherent cells, and loss of membrane integrity. We conclude that the Va phenotype is caused by mutation-induced TRPML3 gain-of-function, resulting in cell death.calcium channel ͉ lysosome ͉ mucolipidosis ͉ TRPML ͉ hair T RPML1 is a putative intracellular ion channel that colocalizes with late endosomal/lysosomal markers (1-3). TRPML2 and TRPML3 are less well understood but are also presumed to be primarily intracellular channels that mediate ion fluxes across endosomal membranes (4). TRPML3 is mutated in varitintwaddler (Va and VaJ) mice (5). Mice homozygous or heterozygous for the Va (A419P) mutation are deaf and exhibit circling behavior indicative of a vestibular defect. Heterozygotes display a variegated and dilute coat color, whereas homozygotes are almost white and have reduced viability (6). A second mutation in TRPML3 arising in the original Va stock (A419P; I362T) results in a less-severe (VaJ) phenotype (7).Mucolipin channel function is unclear because of conflicting data from heterologously expressed, presumed TRPML currents. TRPML1 was claimed to underlie nonselective outwardly rectifying monovalent (8) or proton-conducting (9) currents, but neither of these results were reproduced by others (1). Here, we show that the A419P mutation in the presumed segment (S)4-S5 linker results in a constitutively active, inwardly rectifying cation channel that can be measured across the cell's plasma membrane. Mutation of the corresponding amino acids in TRPML1 results in similar overall conductance properties. WT TRPML3 is highly expressed in melanocytes, but these cells are lost in Va/Va mice, as assessed by the disappearance of melanocyte markers. Consistent with the toxicity of TRPML3Va expression in melanocyte cell lines, we hypothesize that the loss of fur ...

Summary 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-α and EGFR. Conversely, excessive activities of TGF-α/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 Ca2+-permeant channels, forms a signaling complex with TGF-α/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-α release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca2+-dependent cross-linking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.

SummaryTrace metals such as iron, copper, zinc, manganese, and cobalt are essential cofactors for many cellular enzymes. Extensive research on iron, the most abundant transition metal in biology, has contributed to an increased understanding of the molecular machinery involved in maintaining its homeostasis in mammalian peripheral tissues. However, the cellular and intercellular iron transport mechanisms in the central nervous system (CNS) are still poorly understood. Accumulating evidence suggests that impaired iron metabolism is an initial cause of neurodegeneration, and several common genetic and sporadic neurodegenerative disorders have been proposed to be associated with dysregulated CNS iron homeostasis. This review aims to provide a summary of the molecular mechanisms of brain iron transport. Our discussion is focused on iron transport across endothelial cells of the blood-brain barrier and within the neuro-and glial-vascular units of the brain, with the aim of revealing novel therapeutic targets for neurodegenerative and CNS disorders. KeywordsBlood-brain barrier (BBB); Reactive-Oxygen Species (ROS); ferritin (Ft); transient receptor potential mucolipin 1 (TRPML1); transferrin (Tf); non-transferrin-bound iron (NTBI); divalent metal transporter-1 (DMT1, Slc11a2); ferroportin (Fpn); early endosome (EE); brain vascular endothelial cell (BVEC) The Axis of Brain Iron, Oxidative Stress, and NeurodegenerationIron is likely an integral part of metabolism because it can gain (ferric to ferrous, or Fe 3+ to Fe 2+ ) or lose (Fe 2+ to Fe 3+ ) electrons relatively easily. Interestingly, iron has a functional split personality in the nervous system where it is essential for life yet toxic if levels are perturbed. At the cellular level, iron is required for the cell growth, however, excessive iron (iron overload) causes oxidative stress and cell death. Perhaps not surprisingly, iron levels are tightly regulated in a process referred to as iron homeostasis. The principal protective strategy to avoid iron overload in the brain is the blood-brain barrier (BBB), which limits iron entry to the brain from the blood via highly regulated, selective transport systems [1][2][3]. Within the brain, multiple feedback loops form an elaborate control system for cellular iron levels to ensure that a precisely balanced iron level exists for normal function of the nervous system [4,5].*To whom correspondence should be addressed: Haoxing Xu haoxingx@umich.edu. NIH Public Access Author ManuscriptFuture Med Chem. Author manuscript; available in PMC 2010 July 1. Published in final edited form as:Future Med Chem. 2010 January ; 2(1): 51. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIron is critical for a host of basic cellular processes such as mitochondrial ATP generation and DNA replication [6]. Iron deficiency in the brain likely affects normal cell division of brain cells such as neuronal precursor cells, astrocytes, and oligodendrocytes [5,7]. In addition, iron is required for several neuronal specific funct...