Life and Death of Sensory Hair Cells Expressing Constitutively Active
TRPML3 |
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Authors: | Christian Grimm Simone J?rs Stefan Heller |
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Institution: | Departments of Otolaryngology-Head and Neck Surgery and Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5739 |
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Abstract: | The varitint-waddler mutation A419P renders TRPML3 constitutively active,
resulting in cationic overload, particularly in sustained influx of
Ca2+. TRPML3 is expressed by inner ear sensory hair cells, and we
were intrigued by the fact that hair cells are able to cope with expressing
the TRPML3(A419P) isoform for weeks before they ultimately die. We
hypothesized that the survival of varitint-waddler hair cells is linked to
their ability to deal with Ca2+ loads due to the abundance of
plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2
significantly reduced Ca2+]i increase and
apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform
of PMCA2, operating at 30% efficacy, showed a significantly decreased ability
to rescue the Ca2+ loading of cells expressing TRPML3(A419P). When
we combined mice heterozygous for the varitint-waddler mutant allele with mice
heterozygous for the deaf-waddler mutant allele, we found severe hair bundle
defects as well as increased hair cell loss compared with mice heterozygous
for each mutant allele alone. Furthermore, 3-week-old double mutant mice
lacked auditory brainstem responses, which were present in their respective
littermates containing single mutant alleles. Likewise, heterozygous double
mutant mice exhibited severe circling behavior, which was not observed in mice
heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a
molecular rationale for the delayed hair cell loss in varitint-waddler mice.
They also show that hair cells are able to survive for weeks with sustained
Ca2+ loading, which implies that Ca2+ loading is an
unlikely primary cause of hair cell death in ototoxic stress situations.Varitint-waddler (Va) mice express a mutant isoform (A419P) of the
transient receptor potential channel TRPML3 (murine gene symbol,
Mcoln3) that results in profound hearing loss, vestibular defects
(circling behavior, imbalance, head bobbing, waddling), pigmentation
deficiencies, sterility, and perinatal lethality in homozygous animals
(1). A second Mcoln3
variant (VaJ) that arose in the Va background
carries two mutations (I362T and A419P) and shows a phenotype with reduced
severity, particularly in heterozygous animals
(1). The A419P mutation in
Va and VaJ mice is located in
transmembrane-spanning domain
5(TM5)3 of TRPML3,
where it leads to a constitutively open channel, resulting in highly elevated
Ca2+]i
(2-5).
In contrast to the effect of the A419P mutation on TRPML3 channel activity,
the single I362T mutation does not appear to affect
Ca2+]i
(3,
5). When combined with the
A419P mutation, as found in VaJ mice, the constitutive
activity of this mutant TRPML3 isoform is comparable with that of A419P alone
(2-5).Here, we show that HEK293 cells expressing TRPML3-(A419P) or
TRPML3(I362T/A419P) undergo rapid apoptosis. This apoptosis is suppressed by
coexpression of plasma membrane calcium ATPase type 2 (PMCA2). In
varitint-waddler mice, sensory hair cells survive for weeks after birth
(6), which raised the question
of whether this survival could be the result of the hair cells'' ability to
deal with normally transient and localized Ca2+ influx, a feature
that is centered around the high levels of mobile Ca2+ buffers and
PMCA isoforms found in sensory hair cells
(7-10).
We decided to test this hypothesis in vivo by utilizing deaf-waddler
mice that carry a mutation (G283S) in the Atp2b2 gene encoding mutant
PMCA2. Mice homozygous for PMCA2(G283S)
(Atp2b2dfw/dfw) are deaf and have poor
balance (11). Compared with
Atp2b2 knock-out mice, deaf-waddler mice display a milder phenotype
because PMCA2(G283S) retains 30% of its biological activity compared with the
wild-type isoform (12). We
found that sensory hair cell loss, hearing loss, and vestibular dysfunction
were aggravated in mice carrying varitint-waddler and deaf-waddler alleles
compared with animals carrying the single mutant alleles. Our results reveal
that the Ca2+-buffering and Ca2+ extrusion abilities of
hair cells are powerful enough to prevent cell death for weeks, even in the
presence of constitutively active TRPML3(A419P), which is able to induce rapid
apoptosis in other cells. |
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