Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint–waddler mice

Abstract

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 ...

A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype

“…The cells were sequentially perfused with solutions containing 140 or 0 mM Na þ , as indicated by the dark and light grey bars. Almost all TRP channels function as non-selective, Ca 2 þ -permeable channels (Pedersen et al, 2005;Nilius et al, 2007), and TRPML3(A419P) is permeable to Ca 2 þ (Grimm et al, 2007) and conducts Ca 2 þ at isotonic extracellular Ca 2 þ (Ca 2 þ o ) (Xu et al, 2007). Notably, Figures 1D (Owsianik et al, 2006), indicating that TRPML3 is a Ca 2 þ channel that is also permeable to monovalent cations.…”

“…Indeed, the native TRPML3 is found in unidentified vesicular compartments and in the stereocilia outer membrane in the hair cells of the cochlea (Di Palma et al, 2002). All the groups that reported the function of TRPML3(A419P) found that, similar to the native TRPML3, recombinant TRPML3 is expressed at the plasma membrane and intracellular vesicles (Grimm et al, 2007;Kim et al, 2007;Xu et al, 2007;Nagata et al, 2008). The report that TRPML3 is expressed exclusively in the endoplasmic reticulum (Venkatachalam et al, 2006) is probably the result of mistargeting of the channel due to masking of C-terminal targeting motifs (Song et al, 2006).…”

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs

“…In this setting, the extracellular side is equivalent to the luminal side of TRPML1 in endo/lysosomes. The constitutively active V432P mutant, equivalent to the gain-of-function mutation of the mouse TRPML3 in the varitint-waddler ( va ) phenotype 15,27-29 , can be trafficked to the plasma membrane without replacing the di-leucine motifs and therefore V432P and V432P/D472N mutants were both constructed on the background of the wild type channel. The N-terminus truncation mutant was constructed on the background of MmTRPML1-4A.…”

“…Four linker helices surround the S6 bundle crossing and stabilize the closed channel pore. It is interesting to note that several gain-of-function mutations similar to the V432P varitint-waddler phenotype 15,27-29 have been identified on S5 in a proline-scanning mutagenesis study 30 , including R427P, C430P and C431P, which are all clustered at the interaction interface between the S5 and S4-S5 linker (Fig. 4c).…”

“…Among the three isoforms of TRPML channels (TRPML1-3), TRPML1 is ubiquitously expressed in mammalian cells and has been subjected to extensive studies 2,9-14 . TRPML1 is a Ca 2+ -permeable, non-selective, six-transmembrane (6-TM) tetrameric cation channel, and is believed to be the main lysosomal Ca 2+ release channel important for lysosomal trafficking and signal transduction 15-19 . Like most TRP channels, TRPML1 is ligand-gated and can be activated by the endolysosomal specific lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) 7,8 , but is inhibited by the plasma membrane-localized phosphoinositide isoform PI(4,5)P 2 14 (Extended Data Fig.…”