Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles

TRPML3 is an inward rectifying Ca 2+ channel that is regulated by extracytosolic H + . Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca 2+ in the vicinity of cellular organelles that is necessary to regulate these cellular events. TRPML3 is a member of the TRPML subfamily of the transient receptor potential (TRP) channel superfamily (1). The TRPML subfamily was established with identification of TRPML1 as the protein that is mutated in the lysosomal storage disease mucolipidosis type IV (MLIV) (2). Subsequently, TRPML3 was found by positional cloning as the channel mutated in the mouse varitint-waddler phenotype (3), and TRPML2 was identified by database searches (1). The varitint-waddler phenotype is caused by the gain-of-function mutation A419P in TRPML3 (4-8).Members of the TRPML subfamily share the same basic structure of six transmembrane domains, a pore domain between transmembrane domains 5 and 6 and a unique large extracytosolic loop between transmembrane domains 1 and 2.TRPML1 is a lysosomal channel (9) that functions to regulate lysosomal pH (10,11) and thereby membrane trafficking (11,12). TRPML1 was suggested to regulate interaction between late endosomes and lysosomes to control delivery of cellular material to lysosomes (12). However, a recent work used knockdown (KD) of TRPML1 to show that absence of TRPML1 causes loss of protein and lipid hydrolytic activity that led to lysosomal dysfunction (11). Lysosomal dysfunction appears to be associated with increased autophagy (13). TRPML1 can function as a Ca 2+ channel (4). A new potential role for TRPML1 is to be the channel activated by the Ca Much less is known about the localization and function of TRPML3. It was suggested that TRPML3 localizes in the endoplasmic reticulum (ER) when expressed alone, but to be escorted to the lysosomes when coexpressed with TRPML1 or TRPML2 (16). However, the native TRPML3 is expressed in intracellular vesicular compar...

Mutations in the gene Mucolipidosis type IV (MLIV)2 is a lipid storage disorder characterized by an abnormal accumulation of membranous lipids in patients' cells (reviewed in Refs. 1 and 2). Clinically, the disease manifests as corneal clouding, degeneration of the retina, and severe psychomotor retardation (1-6). MLIV is associated with mutations in MCOLN1 (TRP-ML1), a member of the TRP (transient receptor potential) family of ion channels (7-9). The TRP family includes several members that are implicated in human diseases, such as TRPP2 (10), TRPM1 (11), and TRPV6 (12). A critical question in MLIV pathogenesis is why do mutations in TRP-ML1 lead to the cellular phenotype of MLIV?Previous work on the ion selectivity and permeation of TRP-ML1 produced conflicting results. Thus, transient expression in Xenopus oocytes and in fibroblasts suggests that TRP-ML1 is targeted to the lysosomes and functions as a Ca 2ϩ -permeable channel that may regulate lysosomal Ca 2ϩ release and consequently agonist-evoked Ca 2ϩ signals (13,14). On the other hand, TRP-ML1 synthesized in cell-free system and reconstituted into planar lipid bilayers behaves as a monovalent cations permeable, outwardly rectifying channel (15). The outward rectification indicates that when present in lysosomes, TRP-ML1 primarily moves ions into the lysosomal lumen. The outward rectification makes it unlikely that in vivo TRP-ML1 would function as a lysosomal Ca 2ϩ release channel, which suggested an alternative role of TRP-ML1 in lysosomal and cellular functions.In the present report we analyzed the expression pattern and channel properties of TRP-ML1 and several disease-associated mutants. We report that TRP-ML1 is an outwardly rectifying monovalent cationpermeable channel that is primarily expressed in the lysosomes. In the lysosomes, TRP-ML1 is inactivated by proteolytic cleavage. These findings suggest a novel mechanism of regulating TRP-ML1 function.

TRPML3 is a member of the TRPML subfamily of the transient receptor potential cation channel superfamily. The TRPML3(A419P) mutation causes a severe form, whereas the TRPML3(I362T/A419P) mutation results in a mild form of the varitint-waddler phenotype. The channel properties of TRPML3 and how the mutations cause each phenotype are not known. In this study, we report the first channel properties of TRPML3 as a strongly inward rectifying cation channel with a novel regulation by extracytosolic Na ؉ . Preincubating the extracytosolic face of TRPML3 in Na ؉ -free medium is required for channel activation, but then the channel slowly inactivates. The A419P mutation locks the channel in an open unregulated state. Similar gain of function was observed with the A419G mutation, which, like A419P, is expected to destabilize the ␣-helical fifth transmembrane domain of TRPML3. The I362T mutation results in an inactive channel, but the channel properties of TRPML3(I362T/A419P) are similar to those of TRPML3(A419P). However, the surface expression and current density of TRPML3(I362T/A419P) are lower than those of TRPML3(A419P). The A419P mutation also affects channel glycosylation and causes massive cell death. These findings show that the varitint-waddler phenotype is due to a gain of function of TRPML3(A419P) that is reduced by the TRPML3(I362T/A419P) mutant, resulting in a milder phenotype.TRPML3 is a putative channel belonging to the TRPML subfamily of the transient receptor potential channels (1). The TRPML subfamily consists of three members. Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (2, 3). The A419P mutations in the ␣-helical fifth transmembrane domain of TRPML3 cause the varitint-waddler phenotype, which is characterized by pigmentation defect, hearing loss, circling behavior, and embryonic lethality (4, 5). Homozygotes for the A419P mutation have a severe phenotype that results in embryonic lethality. A milder phenotype is observed with the ϩ/A419P heterozygote, when the I362T mutation occurs in the same allele as A419P (4, 5).The channel function of TRPML3 and the effect of the A419P and I362T mutations on channel function are unknown. We report here that TRPML3 functions as an inward rectifying cation channel that is inactivated by extracytosolic cations. The A419P mutation locks the channel in an open state and eliminates regulation of the channel by extracytosolic cations. The I362T mutation inhibits channel activity and reduces the surface expression and current density of the A419P mutant. We conclude that the varitint-waddler phenotype is the result of a gain-of-function mutation in TRPML3. EXPERIMENTAL PROCEDURESPlasmid Construction, Mutagenesis, and Reagents-Human TRPML3 was amplified from human placenta and cloned into the pEGFPC1 and p3XFLAG-CMV-7.1 vectors. Mutations were introduced with the QuikChange kit.Cell Culture, Transfection, and Western Blotting-HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells ...

TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca 2 þ -permeable channel with a unique form of regulation by extracytosolic (luminal) H þ (H þ e-cyto ). Regulation by H þ e-cyto is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H þ e-cyto to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H þ e-cyto , and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H þ e-cyto are altered by both the a-helix-destabilizing A419G and the a-helixfavouring A419M and A419K. These findings suggest that regulation of TRPML3 by H þ e-cyto is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication.