Gain-of-function Mutation in TRPML3 Causes the Mouse Varitint-Waddler Phenotype

Abstract

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 ...

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

“…After incubation in Na þ -free solution, pH 7.4, re-application of Na þ o results in a transient, rapidly inactivating current by TRPML3(H283R), which is inhibitable by H þ e-cyto . These TRPML3(H283A) properties are the same as those reported for TRPML3(A419P) (Kim et al, 2007). To further compare between the two mutants, we measured their effect on cell survival and plasma membrane Ca 2 þ permeability.…”

“…Recently we (Kim et al, 2007) and others (Grimm et al, 2007;Xu et al, 2007;Nagata et al, 2008) reported that the varitintwaddler phenotype is caused by a GOF that makes TRPML3(A419P) spontaneously active, as found with TRPML3(H283A). Moreover, both mutants similarly increase plasma membrane Ca 2 þ permeability and reduce cell survival in a Ca 2 þ -dependent manner ( Figure 3E and F).…”

“…Recently, we reported that TRPML3 functions as an inwardly rectifying cation channel and that channel activity requires incubation in Na þ -free media (Kim et al, 2007). In this study, we sought to determine the mechanism of this effect.…”

“…We have recently reported that TRPML3 functions as an inwardly rectifying cation channel, and that the A419P mutation, which causes the varitint-waddler phenotype, is a gainof-function mutation (GOF) (Kim et al, 2007). Similar GOF by TRPML3(A419P) was reported by three additional studies (Grimm et al, 2007;Xu et al, 2007;Nagata et al, 2008), although these studies did not examine the properties of wild-type TRPML3 or its regulation.…”

“…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).…”