Abstract
The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca(2+). 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 Ca(2+) loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca(2+)](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 Ca(2+) 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 Ca(2+) loading, which implies that Ca(2+) loading is an unlikely primary cause of hair cell death in ototoxic stress situations.
Highlights
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
TRPML3(A419P)-mediated Cell Death Is Reduced by Coex- to the cell surface (Fig. 2, A and B), which is an early diagnostic pression of plasma membrane calcium ATPase type 2 (PMCA2)—Constitutively active TRPML3(A419P) sign of apoptotic cell death that can be revealed with annexin severely affects cell morphology and survival of LLC-PK1-CL4 V staining
We investigated the mechanism by which sensory hair cells are able to deal with a severe cationic overload caused by constitutive activity of the TRPML3 channel in varitint-waddler mice
Summary
Plasmid Constructs—The cDNAs encoding wild-type and mutant TRPML3 channel isoforms [3] were subcloned into pcDNA3.1 (Invitrogen). Rat PMCA2z/a cDNA plasmid [13] was provided by Dr Peter G. Gillespie (Oregon Hearing Research Center, Portland, OR).
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