Life and death of sensory hair cells expressing constitutively active TRPML3

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.

FIGURE 1.

HEK293, NIH3T3, and HeLa cells expressing either wild-type TRPML3 or the TRPML3(A419P) varitint-waddler mutant isoform. Shown are representative cells expressing the respective C-terminal yellow fluorescent protein fusion constructs of wild-type TRPML3 and mutant TRPML3 15 h after transfection. Scale bars = 20 μm.

FIGURE 2.

Survival of HEK293 cells expressing varitint-waddler mutant isoforms of TRPML3 with or without PMCA2. A and B, percentage of annexin V-positive cells at various time points (10, 15, 20, and 25 h) after cotransfection of varitint-waddler mutant isoforms of TRPML3 (C-terminally fused to yellow fluorescent protein (YFP)) with rat PMCA2 or empty vector (pcDNA3.1). Coexpression of wild-type (Wt) TRPML3 with PMCA2 or the I362T mutant isoform of TRPML3 with PMCA2 did not show any annexin V-positive staining after 25 h. These experiments were conducted in the context of a previous publication; hence, the control data shown here (+ pcDNA3.1) have been published previously (3). ^***, p < 0.0001; ^**, p < 0.001; ^*, p < 0. 01 (Student's t test, unpaired).

FIGURE 3.

Calcium imaging of HEK293 cells expressing varitint-waddler mutant isoforms of TRPML3 with or without PMCA2. A and B, Ca²⁺ imaging results showing relative [Ca²⁺]_i of HEK293 cells coexpressing wild-type (Wt) TRPML3, the I362T mutant isoform, or varitint-waddler isoforms in combination with PMCA2, the PMCA2 deaf-waddler mutant isoform (G283S), or empty vector (pcDNA3.1). All expression vectors were cotransfected at molar ratios of 1:1. Values are the means ± S.E. (n shown in parentheses). All measurements were performed 15 h after transfection. C, Ca²⁺ imaging experiments as described for A and B using 1 mm sodium vanadate to block the PMCA2 effect. Cultured cells were preincubated with 1 mm sodium vanadate for 4 h before measuring. ^***, p < 0.0001; ^**, p < 0.001; ^*, p < 0. 01 (Student's t test, unpaired); n.s., not significant. Mean values are the result of n independent measurements with 10-20 cells each. All mean values were corrected for the values of non-transfected control cells in each measurement.

FIGURE 4.

Constitutively active conductance elicited by varitint-waddler TRPML3 mutants in HEK293 cells in the presence and absence of PMCA2. A and B, average inward current densities at -80 mV of the murine A419P and I362T/A419P mutants, respectively, in the presence of rat PMCA2 compared with cotransfections with empty vector cDNA (pcDNA3.1), normalized by cell capacitance (picofarads (pF)). All expression vectors were cotransfected at molar ratios of 1:1. n is the number of cells analyzed. C and D, steady-state current-voltage plots of whole-cell currents elicited in cells cotransfected with the murine A419P and I362T/A419P mutants, respectively, and rat PMCA2 or empty vector cDNA in response to 10-ms voltage steps from a holding potential of +10 mV between -200 and +100 mV in 20-mV incremental steps, normalized by cell capacitance. E and F, same plot but normalized to maximal current elicited at -200 mV to demonstrate similarity in responses.

FIGURE 5.

Genotyping and analysis of cochlear hair cell degeneration in P10 and P21 varitint-waddler, deaf-waddler, and varitint-waddler/deaf-waddler double mutant mice. A, genotyping results showing genomic DNA sequence traces of wild-type mice, mice heterozygous for Va (Mcoln3^Va/+), and mice hetero- and homozygous for deaf-waddler (Atp2b2^dfw/+ and Atp2b2^dfw/dfw), as well as DNA sequences of double mutant animals (Mcoln3^Va/+;Atp2b2^dfw/+ and Mcoln3^Va/+;Atp2b2^dfw/dfw). Respective DNA sequence changes take place at position 1255 (G → C) of Mcoln3 (varitint-waddler) and at position 847 (G → A) of Atp2b2 (deaf-waddler). B, hair cell bundle degeneration in P10 and P21 mice as genotyped in A. Shown are IHCs and OHCs from apical whole-mount preparations labeled with phalloidin (F-actin) and anti-parvalbumin 3 antibody (PV3). Scale bars = 10 μm. C, quantification of hair cell (HC) numbers at P10. D, quantification of hair cell numbers at P21. Shown are the average numbers of IHCs and OHCs in the apical part of the cochlea (hair cells/100 μm, means ± S.E. of at least three independent samples each). ^***, p < 0.0001; ^**, p < 0.001; ^*, p < 0. 01 (Student's t test, unpaired).

FIGURE 6.

ABR measurements. A, shown are representative ABRs of 3-week-old wild-type (Wt; RSV/LeJ), heterozygous varitint-waddler (TRPML3^Va/+), heterozygous deaf-waddler (PMCA2^dfw/+), and varitint-waddler/deaf-waddler double mutant (TRPML3^Va/+;PMCA2^dfw/+) mice to a click stimulus. SPL, sound pressure level. B, shown are ABR thresholds (means ± S.E.) to click and 8-, 16-, and 32-kHz stimuli (n = number of animals).

FIGURE 7.

Analysis of vestibular hair cell degeneration in varitint-waddler, deaf-waddler, and varitint-waddler/deaf-waddler double mutant mice. A and B, representative immunofluorescence stainings of P21 murine utricle preparations. The higher magnification examples (B) are from comparable areas in the striola region, where the phenotype was most noticeable. Preparations were stained with phalloidin (F-actin) and anti-calretinin antibody. Asterisks indicate hair cells without bundles. Scale bars = 100 μm (A) and 20 μm (B). C, percentage of hair cells (HC) without bundles at P21 in the striola region of the utricle (hair cells/50 μm², means ± S.E. of at least three independent samples each). ^***, p < 0.001; ^**, p < 0.01; ^*, p < 0. 05 (Student's t test, unpaired).

FIGURE 8.

Circling behavior of varitint-waddler/deaf-waddler double mutant mice. The quantification was conducted at 3 and 6 weeks postnatal (P21 and P42, respectively). Wild-type littermates (RSV/LeJ), heterozygous varitint-waddler mice (Mcoln3^Va/+;Atp2b2^+/+), and PMCA2(G283S) mutant mice (Mcoln3^+/+;Atp2b2^dfw/+ and Mcoln3^+/+;Atp2b2^dfw/dfw) did not display circling behavior at P21 and P42 (n = number of mice tested). 67% (n = 6/9, P21) and 71% (n = 12/17, P42) of the heterozygous double mutants (Mcoln3^Va/+;Atp2b2^dfw/+) and 100% (n = 9/9, P21; n = 6/6, P42) of the Mcoln3^Va/+;Atp2b2^dfw/dfw double mutants showed severe circling behavior. Non-circling mice were excluded from rpm calculations. For each mouse, we quantified circling of three tests that were performed at consistent time points on different days.