The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration

Abstract

Varitint-waddler (Va andVaA very common cause of deafness is the loss of hair cells, which degenerate because of environmental factors or genetic mutations. Varitint-waddler (Va) mice have a mutation that causes an alanine-to-proline substitution (A419P) in the fifth transmembrane domain of TRPML3, a presumed ion channel. A second allele, Va J , contains the A419P mutation in cis to an I362T mutation (1). The earliest sign of inner ear damage in Va mice is hair cell degeneration, which begins in embryogenesis as hair cells differentiate and continues postnatally. During degeneration, the hair cells bulge out of the apical side of the epithelium and become extruded. Eventually these mice also develop abnormalities in supporting cells, in the tectorial membrane, and in the stria vascularis and in the endocochlear potential that they help generate. The defects of Va J mice are similar although less severe, suggesting that the mutation I362T attenuates the effects of A419P (1-3). A comprehensive examination of TRPML3 expression in inner ear has not been published. Expression of TRPML3 protein in hair cells was suggested by immunocytochemical methods, although results for other inner ear cell types were not reported (1).Mutations in two other TRP channels, Drosophila TRP and human TRPML1 (mutations in which cause mucolipidosis type IV), are associated with degeneration of the retina. In both cases, the channels seem necessary for cellular viability, because recessive, loss-of-function mutations cause degeneration (4, 5).By contrast, the Va and Va J mutations in TRPML3 are semidominant, although it remains unclear whether this is due to gain-of-function, haploinsufficiency, or dominant-negative effects (1, 6). ResultsWe first established which cells expressed TRPML3 in the inner ear of wild-type mice. In situ hybridization on mouse inner ear using probes from two nonoverlapping regions of TRPML3 mRNA gave identical results [ Fig. 1 and supporting information (SI) Fig. 6]. TRPML3 mRNA was most abundant in the marginal cells of the cochlear stria vascularis and the dark cells of the vestibule (Fig. 1 i, l, and m and SI Fig. 6 i, l, and m). Both cell types are involved in producing the endolymph and therefore the endocochlear potential (7-9). This expression pattern is in keeping with the reduced stria vascularis and the rounding up and loss of the cytoplasmic processes of its marginal cells in Va/Va and Va/ϩ mice (2) and with the reduced endocochlear potentials of the Va J mutants (1, 3).In addition, other cells that line the cochlear scala media and the connected vestibular endolymphatic spaces expressed TRPML3 mRNA, including (i) supporting cells at the marginal part of the lesser epithelial ridge (Hensen and Claudius cells), which in Va and Va J mice (homozygotes and heterozygotes) appear undifferentiated or degenerate ( Fig. 1i and SI Fig. 6i); (ii) cells of the spiral limbus that produce the tectorial membrane, which in Va mice develops abnormally and fails to attach to the reticular lamina ( Fig. 1i and SI Fig. 6i...

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).…”

A Quantitative Analysis of the Spatiotemporal Pattern of Transient Receptor Potential Gene Expression in the Developing Mouse Cochlea

“…Although TRPML3 is expressed in the inner ear (Nagata et al 2008;Van Aken et al 2008;Grimm et al 2009;Takumida and Anniko 2009), our expression screen did not reveal large changes in expression between E17 and P8. TRPML3 showed a shallow RNA expression gradient during development and across the organ.…”

“…TRPV4 is expressed in hair cells, stria vascularis, and vestibular dark cells (Liedtke et al 2000;Takumida et al 2005), and disruption of TRPV4 causes delayed-onset hearing loss and increases susceptibility to acoustic injury (Tabuchi et al 2005). TRPML3 is expressed in sensory hair cells and in the marginal cells of the stria vascularis (Di Palma et al 2002;Nagata et al 2008;Van Aken et al 2008). Mutations in TRPML3 lead to deafness in varitintwaddler (Va) mice due to defects in stria vascularis and hair cells.…”

“…Mutations in TRPML3 lead to deafness in varitintwaddler (Va) mice due to defects in stria vascularis and hair cells. The Va mutation induces constitutive activation of inward currents which causes cellular degeneration (Nagata et al 2008;Van Aken et al 2008).…”