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.

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...

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 Fe 3+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 Fe 2+ transporter currently known and is highly expressed in erythroid precursors 6, 9 , but genetic studies suggest the existence of a DMT1-independent endosomal/lysosomal Fe 2+ 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 Fe 2+ permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair TRPML1's ability to permeate Fe 2+ at varying degrees, which correlate well with the disease severity. A comparison of TRPML1 −/− ML4 and control skin fibroblasts showed a reduction of cytosolic Fe 2+ levels, an increase of intra-lysosomal Fe 2+ levels, and an accumulation of lipofuscin-like molecules in TRPML1 −/− cells. We propose that TRPML1 mediates a mechanism by which Fe 2+ is released from late endosomes/lysosomes. Our results suggest that impaired iron transport may contribute to both hematological and degenerative symptoms of ML4 patients.

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 gainof-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 Ca 2؉ -dependent, constitutive Ca 2؉ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca 2؉ 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, protonimpermeable, Ca 2؉ and Fe 2؉/Mn 2؉ 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.

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