Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.

SUMMARYIn peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/-mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.

Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitintwaddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembranespanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.cation channel ͉ cochlea ͉ hair cell ͉ inner ear ͉ TRP channel T ransient receptor potential (TRP) cation channels are important components of many cellular and sensory systems, such as osmosensation, photosensation, taste sensation, and thermosensation (1, 2). In Drosophila, Caenorhabditis elegans, and lower vertebrates, mutations in TRP channels have also been associated with loss of sensitivity to touch and other forms of mechanical stimulation (3-5). Besides the general notion that they conduct cations, TRP channels have quite diverse structural and functional properties (1, 2).The identification of mutations in the human mucolipin (TRPML1) gene as the cause of mucolipidosis type IV, a neurodegenerative, recessive, lysosomal storage disorder characterized by psychomotor retardation and visual impairment, led to the initial description of the TRPML subfamily (6, 7). The mammalian TRPML subfamily consists of three members: TRPML1, TRPML2, and TRPML3. Like other TRP channels TRPML proteins contain six putative transmembrane domains with cytosolic amino and carboxyl termini. A mutant isoform of murine Trpml3 has recently been identified as the underlying cause of the varitint-waddler (Va) phenotype that is manifested in profound deafness, vestibular defects, pigmentation deficiencies, sterility, and perinatal lethality (8, 9). The Trpml3 (Va) allele displays an alanine to proline substitution at amino acid position 419 (A419P) in the fifth transmembrane domain of TRPML3 (8) [supporting information (SI) Fig. 5]. A second amino acid substitution (I362T) positioned at the second extracellular loop arose in cis to A419P, resulting in the Va J allele that attenuates and partially rescues the severe Va phenotype by an unknown mechanism.The present work was conducted to determine the specific role of the varitint-waddl...

Summary We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na+]. This synergism revealed existence of distinct and cooperative activation mechanisms, and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are non-responsive to the activators identified in this screen.