Genetic inactivation of Trpml3 does not lead to hearing and vestibular impairment in mice.

Abstract

TRPML3, a member of the transient receptor potential (TRP) family, is an inwardly rectifying, non-selective Ca2+-permeable cation channel that is regulated by extracytosolic Na+ and H+ and can be activated by a variety of small molecules. The severe auditory and vestibular phenotype of the TRPML3(A419P) varitint-waddler mutation made this protein particularly interesting for inner ear biology. To elucidate the physiological role of murine TRPML3, we conditionally inactivated Trpml3 in mice. Surprisingly, lack of functional TRPML3 did not lead to circling behavior, balance impairment or hearing loss.

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Genetic Inactivation of Trpml3 Does Not Lead to Hearing
and Vestibular Impairment in Mice
Simone Jo ¨rs., Christian Grimm., Lars Becker, Stefan Heller*
Departments of Otolaryngology – Head and Neck Surgery and Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, United
States of America
Abstract
TRPML3, a member of the transient receptor potential (TRP) family, is an inwardly rectifying, non-selective Ca2+-permeable
cation channel that is regulated by extracytosolic Na+and H+and can be activated by a variety of small molecules. The
severe auditory and vestibular phenotype of the TRPML3(A419P) varitint-waddler mutation made this protein particularly
interesting for inner ear biology. To elucidate the physiological role of murine TRPML3, we conditionally inactivated Trpml3
in mice. Surprisingly, lack of functional TRPML3 did not lead to circling behavior, balance impairment or hearing loss.
Citation: Jo ¨rs S, Grimm C, Becker L, Heller S (2010) Genetic Inactivation of Trpml3 Does Not Lead to Hearing and Vestibular Impairment in Mice. PLoS ONE 5(12):
e14317. doi:10.1371/journal.pone.0014317
Editor: Hiroaki Matsunami, Duke University, United States of America
Received July 12, 2010; Accepted November 21, 2010; Published December 13, 2010
Copyright: ? 2010 Joers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants DC004563 and P30 DC010363. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hellers@stanford.edu
. These authors contributed equally to this work.
Introduction
The mammalian TRPML gene family consists of TRPML1,
TRPML2, and TRPML3. Mutations in human TRPML1 cause
the lysosomal storage disease Mucolipidosis Type IV [1–3]. The
murine Trpml2 and Trpml3 genes were identified in a positional
cloning approach to find the gene responsible for the varitint-
waddler (Va) phenotype [4]. Heterozygote Va mice have
pigmentation defects, hearing loss, and circling behavior, whereas
homozygotes display perinatal lethality [4–6]. The Va phenotype is
caused by a single mutation (A419P) in the predicted fifth
transmembrane-spanning domain (TM5) of TRPML3, which
leads to a constitutively open channel, resulting in elevated [Ca2+]i
causing apoptosis [7–11]. Heterologously expressed TRPML3 is
regulated by extracytosolic Na+and H+[8,12,13] and can be
activated by a variety of small molecules [13]. Subcellular
localization studies of heterologously expressed TRPML channels
revealed that TRPML1 and TRPML2 are expressed in late
endosomes and lysosomes, whereas TRPML3 presumably shuttles
between multiple intracellular compartments and the plasma
membrane [14]. The localization to intracellular vesicles and the
interaction with a variety of vesicular proteins suggest that all three
members may play a role in endocytic and exocytic signalling
pathways [15]. In addition, heterologously expressed TRPML
protein subunits are able to heteromerize with each other [13,16],
and it has been hypothesized that plasma membrane-localized
TRPML3 in epidermal melanocytes occurs exclusively as a
subunit of uncharacterized heteromeric channels [13].
TRPML3 expression has been detected in inner ear sensory
hair cells [4,17], and the Va mutation of the channel leads to hair
cell degeneration and deafness [4,5]. On the other hand, hair cell
death-mediated deafness (due to a constitutive active ion channel)
is not sufficient to justify a function in the hearing process itself. To
elucidate a relevant function for TRPML3 in hair cells, we
generated and examined a mouse model that allows conditional
Trpml3 inactivation. Here we show that ubiquitous as well as hair
cell-specific postnatal inactivation of Trpml3 does not result in
hearing and balance impairment when compared with control
littermates.
Results and Discussion
Generation of a floxed Trpml3 allele (Trpml3lox)
TRP channel knockouts have successfully been generated
through targeted deletion of a genomic region encoding the
presumptive pore-loop domain of the ion channel [18–20].
Therefore, we decided to target exon 11 of Trpml3, which encodes
the pore-loop and the pore lining TM6 (Fig. 1A). We generated a
targeting vector that, after homologous recombination, resulted in
a modified Trpml3 allele carrying two loxP sites flanking exon 11
(Fig. 1B). A neoRcassette, which was used for G418/geneticin
selection, was removed with Flp recombinase before the ES cells
were injected into host blastocysts to generate chimeric mice
(Fig. 1C). After germline transmission and continued breeding,
PCR with genomic DNA from progeny of wild-type, heterozygous
and homozygous animals showed proper recombination and
inheritance of the Trpml3loxallele (Fig. 2A,B). Exon 11 of the
Trpml3loxallele and the genomic sequences surrounding the
recombination site were verified by sequencing.
Excision of Trpml3 exon 11
To investigate whether TRPML3 plays a role in development,
we used the X-linked HprtCre/+driver, which mediates ubiquitous
and highly efficient excision that is complete at the first stages of
development [21]. HprtCre/+;Trpml3lox/+females were mated with
Trpml3lox/+males. The floxed exon 11 was always excised
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regardless of HprtCreinheritance. Oocytes of HprtCrefemales have
sufficiently stored Cre recombinase to excise floxed DNA segments
already at the zygote or early stages [21], which most likely
resulted in Trpml3 inactivation already in the fertilized egg. The
resulting Trpml3Dmutation was stably inherited (Fig 2C–E).
To examine whether the Trpml3Dallele is being transcribed,
RT-PCRs were conducted (Fig. 2D,E). mRNA from kidney and
inner ear was purified from dissected organs of mice of each
genotype. RT-PCR amplification of specific cDNA sequences
before and after the site of deletion indicated that wild-type and
Trpml3DmRNA was expressed in kidney and the inner ear of all
animals (Fig. 2D,E; RT-2 and 3). Amplification of cDNA encoding
exon 11 (RT-1), however, was only possible from mRNA of wild-
type (Trpml3+/+) and heterozygous animals (Trpml3D/+), but not
from mRNA of Trpml3D/Dmice.
An additional oligonucleotide pair was used (RT-4) to amplify
the coding region comprising exons 10, 11, and 12. Sequence
analysis revealed that in mutated animals, splicing occurred
between exon 10 and exon 12, resulting in a 207 bp shorter
message encoding a protein lacking 69 amino acids of the pore
loop and TM6, but without frame shift. To demonstrate that a
pore-less TRPML3 protein does not function as an ion channel,
we generated cDNA encoding a fusion protein of TRPML3(D
exon11) with yellow fluorescent protein and expressed the mutant
channel protein in HEK293 cells. The subcellular distribution of
the mutant channel TRPML3(D exon11), analyzed by confocal
Figure 1. Targeting strategy for disruption of the Trpml3 gene. A, Transmembrane-spanning domains are depicted as grey bars and
numbered from 1–6. The orange frame indicates the part of the TRPML3 protein that is encoded by exon 11, which will be deleted (pore loop and
TM6). Exons are shown as black and orange bars on the schematic genomic map below. B, Shown are the targeting vector and the targeted allele
after homologous recombination. The blue bar represents the PGK promoter-driven neoRexpression cassette, which was used for positive selection.
The DTA cassette, used for negative selection, is shown in green. The black and red arrowheads symbolize position and orientation of FRT and loxP
sites. C, Targeted allele after Flp site-specific recombination in ES cells, resulting in excision of neoRcassette, and leaving one FRT site behind.
D, Excision of exon 11 using Cre site-specific recombinase, resulting in disruption of Trpml3 gene.
doi:10.1371/journal.pone.0014317.g001
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Figure 2. Genotyping analysis. A, Schematic drawing of the Trpml3 targeted allele before (Trpml3lox/) and after Cre recombination (Trpml3D/). The
positions of sense (sn) and antisense (asn) oligonucleotides are indicated with arrows. The names of PCR products and the corresponding lengths are
parenthesized. B, PCR amplification products of Trpml3 locus of homozygous (lox/lox), heterozygous (lox/+), and wild-type (+/+) Trpml3lox/mice are
shown: lox: 309 bp and/or 471 bp, neo: 374 bp, and left arm (LA): 3176 bp and right arm (RA): 3298 bp. C, Genotyping after Cre recombination of
representative HprtCre/;Trpml3D/mice. All mice are heterozygous for cre (210 bp). The same sets of oligonucleotides were used as in (B), however lox-
PCR only displayed the wild-type 309 bp PCR fragment, since the lox asn-oligonucleotides cannot hybridize after the targeted exon 11 was excised;
and RA: 2340 bp, since exon 11 was excised by Cre. The last panel on the right shows the shortened fragment (indicated with a red arrow): 1952 bp
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microscopy, was different from wild-type TRPML3-YFP fusion
protein. Mutant channel protein appeared to be absent from the
plasma membrane and was mainly located intracellularly (Fig. 3A).
When we used the TRPML3 agonist SN-2 to activate TRPML3(D
exon11) [13], we detected no change of [Ca2+]i, whereas wild-type
TRPML3-expressing cells responded with a robust increase in
[Ca2+]i (Fig. 3B). The average maximum [Ca2+]i levels were
0.8160.073(Dratio340nm/380nm,
TRPML3 and 20.01660.009 (Dratio 340nm/380nm, n=6) for
TRPML3(D exon11); the latter measurements did not differ
significantly from the baseline average ratio of 0.31960.038
(n=6), indicating that TRPML3(D exon11) is an inactive ion
channel when expressed in HEK293 cells. Based on these
experiments, we presume that Trpml3
TRPML3 protein.
n=10) forwild-type
D/Dmice lack functional
Evaluation of Balance and Auditory Function
Based on the Va phenotype, TRPML3 has been proposed to
play a role in hearing and balance and the channel has been put
forward as potential candidate for the hair cell mechanoelectrical
transduction channel [22]. Consequently, we were curious to find
out whether Trpml3 inactivation would result in hearing and
balance defects. Three-week-old HprtCre/+;Trpml3+/+, HprtCre/+;
Trpml3D/+, and HprtCre/+;Trpml3D/Dmice had normal Preyer’s
reflexes, characterized by a distinct movement of the pinna in
response to a loud sound [23].
Auditory-evoked brainstem response (ABR) measurements were
used to evaluate hearing thresholds of three-week-old mice (Fig. 3).
Because the Preyer’s reflex and other subjective measures to assess
hearing are only effective for identification of profound hearing
loss, more objective electroacoustical tests were conducted: Click,
8-, 16-, and 32 kHz tone burst measurements revealed no
significant differences in ABR thresholds and interwave latencies
among all three groups: HprtCre/+;Trpml3+/+, HprtCre/+;Trpml3D/+,
and HprtCre/+;Trpml3D/D(Fig. 3A,B). Interwave latencies between
wave I and wave III at 70 dB, which are indicative of the afferent
auditory nerve conductance were 1.960.026 msec in HprtCre/+;
Trpml3+/+animals (n=6), compared to 1.8760.026 msec in
HprtCre/+;Trpml3
Trpml3
TRPML3 is not essential for hair cell function, synaptic signal
transmission, performance of spiral ganglion neurons, and
auditory nerve function. Nevertheless, because the inactivation
of Trpml3 using HprtCre–mediated recombination was introduced
very early in development, compensation effects cannot be
excluded with this approach.
With the goal to circumvent potential compensatory mecha-
nisms, we used tamoxifen-inducible Math1-CreERTMmice [24] to
inactivate TRPML3 in cochlear hair cells between P0–P3. ABR
measurements of these mice at three weeks of age revealed no
differences (Fig. 3C), suggesting that compensatory mechanisms
most likely do not explain the lack of a TRPML3 hearing
phenotype. Mice were also analyzed at 3 months of age to
determine possible enhancement or early onset of age-related
hearing loss. But reviewing audiograms revealed no ABR
threshold difference between mutant mice and control mice (data
not shown). These experiments have two important limitations.
First, the tamoxifen-inducible recombination in cochlear hair cells
is not complete, as revealed by analysis of crosses of Math1-
CreERTMmice with Rosa26-lacZ reporter mice [24]. Therefore, a
substantial number of cochlear hair cells might have been
unaffected by Cre-mediated recombination. The fact that mutant
TRPML3 protein is being expressed (Fig. 3A) complicates the
analysis of Cre-mediated recombination because the mutant pore-
less TRPML3 protein is likely to be detectable by immunohisto-
chemistry. Second, three weeks of loss of TRPML3 function might
be enough for potential compensatory mechanisms to become
effective. Nevertheless, as long as there are no clear candidates or
D/+(n=6), and 1.8660.019 msec in HprtCre/+;
D/Dlittermates (n=6). These results suggested that
for Trpml3lox/and 993 bp for Trpml3D/. D and E, RT-PCR analysis of TRPML3 mRNA expression in kidney and inner ear from 3-week-old HprtCre/;Trpml3D/
mice. D, Schematic illustrationisshowingexon 6–12.Thesense(sn) andantisense (asn)oligonucleotides areindicatedwithblackarrows. E,Amplification
products RT-1(200 bp), RT-2 (512 bp), RT-3 (181 bp), andRT-4 (473 bp for wild-type cDNA, and266 bp for Cretargeted Trpml3 cDNA).Oligonucleotides
for GAPDH (442 bp) were used to control for RNA preparation quality. The symbol Dindicates the deleted exon 11 in the Trpml3 allele generated by
recombination of the floxed (loxP) locus; +, represents the wild-type locus.
doi:10.1371/journal.pone.0014317.g002
Figure 3. Molecular and functional assessment of mutant TRPML3(D exon11) channels. A, Shown are representative cells expressing the
respective murine (m) constructs of wild-type TRPML3 or mutant TRPML3(D exon11), C-terminally fused to yellow fluorescent protein, 24 h after
transfection. Scale bar=10 mm. B, Ca2+imaging results showing relative [Ca2+]iincreases after application of TRPML3 activator SN-2 in HEK293 cells
expressing wild-type TRPML3 or mutant TRPML3(D exon11). Shown are mean values 6 SEM (numbers in parentheses are the numbers of
independent experiments with 10–20 cells each). Statistical comparisons of means were made using Student’s t test (unpaired); ***p,0.0001.
doi:10.1371/journal.pone.0014317.g003
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mechanisms known that could provide compensation for inacti-
vation of Trpml3, it is difficult to speculate about the timing of
compensatory mechanisms. In summary, the use of Math1-
CreERTMmice did not reveal potential compensatory mechanisms
of Trpml3 inactivation, but because of the limitations of this
experiment, we cannot exclude that loss of TRPML3 function is
being compensated by an unknown mechanism.
To investigate whether acoustic challenge of the auditory system
would reveal a more subtle role of TRPML3, we exposed HprtCre/+;
Trpml3+/+and HprtCre/+;Trpml3D/Dmice for 4 hr to 4 kHz pure
tone at 125 dB SPL [25,26]. We compared ABR thresholds of
littermates of both genotypes before and one week after the noise
exposure, and we detected no difference in noise susceptibility
between the two groups (Fig. 4D). Unlike knockout of TRPV4, an
ion channel expressed by cochlear hair cells, spiral ganglion
neurons, and stria vascularis marginal cells [27], which displays
increased susceptibility to acoustic injury [25], mice carrying two
inactive Trpml3 alleles did not show increased acoustic vulnerability.
Besides evaluating the auditory system, we also assessed
vestibular function in HprtCre/+;Trpml3+/+and HprtCre/+;Trpml3D/D
Figure 4. ABR measurements. A, Graph shows representative ABR waveforms of 3-week-old HprtCre/+; Trpml3+/+and HprtCre/+; Trpml3D/Dmice in
response to a click stimulus. ABRs were recorded at sound stimulation intensities of 25–70 dB. ABR waves I–V are indicated above the peaks. Red
arrow highlights the hearing threshold, which is at 35 dB in this representative example pair. B, Shown are ABR thresholds (mean values 6 SEM) to
click, 8-, 16-, and 32 kHz stimuli of HprtCre/+;Trpml3+/+(n=6), HprtCre/+;Trpml3D/+(n=6), and HprtCre/+;Trpml3D/D(n=6) and C, of Math1-CreERCre/+;
Trpml3+/+(n=3), Math1-CreERCre/+;Trpml3D/+(n=3), and Math1-CreERCre/+;Trpml3D/D(n=3), respectively. Statistical comparisons of means of different
genotypes were made using one-way ANOVA followed by Tukey’s post test; p.0.05. D, ABR threshold shifts of 3-month-old HprtCre/+;Trpml3+/+(n=7)
and HprtCre/+;Trpml3D/D(n=7) 1 week after the acoustic overexposure of 125 dB SPL at 4 kHz for 4 hr. Shown are mean values 6 SEM. No significant
differences were observed between genotypes (p.0.05, one-way ANOVA, followed by Tukey’s post test).
doi:10.1371/journal.pone.0014317.g004
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