Auditory mechanotransduction in the absence of functional myosin-XVa

Abstract

In hair cells of all vertebrates, a mechanosensory bundle is formed by stereocilia with precisely graded heights. Unconventional myosin-XVa is critical for formation of this bundle because it transports whirlin and perhaps other molecular components responsible for programmed elongation of stereocilia to the stereocilia tips. A tip of a stereocilium is the site of stereocilia growth and one of the proposed sites of mechano-electrical transduction. In adult shaker 2 mice, a mutation that disables the motor function of myosin-XVa results in profound deafness and abnormally short stereocilia that lack stereocilia links, an indispensable component of mechanotransduction machinery. Therefore, it was assumed that myosin-XVa is required for proper formation of the mechanotransduction apparatus. Here we show that in young postnatal shaker 2 mice, abnormally short stereocilia bundles of auditory hair cells have numerous stereocilia links and 'wild type' mechano-electrical transduction. We compared the mechanotransduction current in auditory hair cells of young normal-hearing littermates, myosin-XVa-deficient shaker 2 mice, and whirler mice that have similarly short stereocilia but intact myosin-XVa at the stereocilia tips. This comparison revealed that the absence of functional myosin-XVa does not disrupt adaptation of the mechanotransduction current during sustained bundle deflection. Thus, the hair cell mechanotransduction complex forms and functions independently from myosin-XVa-based hair bundle morphogenesis.

Figure 2. Intact mechano-electrical transduction in young Myo15^sh2/sh2 OHCs

A, organ of Corti explant with a piezo-driven probe (left) and a patch pipette (right). B and C, typical SEM images of OHC stereocilia bundles at the end of the first (apical) cochlear turn in Myo15^sh2/sh2 (B) and Myo15^+/sh2 (C) organ of Corti explants. D and E, representative whole-cell currents (top traces) evoked by the graded deflections of stereocilia at a holding potential of −90 mV in Myo15^sh2/sh2 (D) and Myo15^+/sh2 (E) mice. Mechanotransduction current rapidly reaches a peak (black arrows) and then decays on a fast and then slow (open arrows) time scale. Responses to the inhibitory bundle deflections are shown in bold. Relatively large access resistances (D −74 MΩ, E −73.5 MΩ) may somewhat decrease the driving force and maximum amplitude of transduction current. F, average relationships between peak transduction current and probe displacement in Myo15^+/sh2 (○, n = 7) and Myo15^sh2/sh2 (•, n = 7) OHCs. Data were fitted with a second order Boltzmann function: I = I_max/[1 + exp(α₂*(p₂ −Δx))*(1 + exp(α₁*(p₁ −Δx)))] − I_min. Parameters of the fit (Myo15^+/sh2/Myo15^sh2/sh2): I_max = 285/240 pA; I_min = 14.4/6.9 pA; α₁ = 12.6/11.7 μm⁻¹; p₁ = 0.11/0.18 μm; α₂ = 4.5/3.7 μm⁻¹; p₂ = 0.28/0.18 μm. Access resistances (Myo15^+/sh2/Myo15^sh2/sh2): 53 ± 6/51 ± 4 MΩ. G, normalized current-displacement relationships in OHCs of Myo15^+/sh2 and Myo15^sh2/sh2 mice at −90 mV holding potential. The averages of individual normalized current-displacement curves are shown. Parameters of the fit (Myo15^+/sh2/Myo15^sh2/sh2): α₁ = 10.7/10.5 μm⁻¹; p₁ = 0.14/0.18 μm; α₂ = 3.8/3.6 μm⁻¹; p₂ = 0.26/0.19 μm. The same OHCs contributed to F and G. Vertical bars indicate Standard Errors. Scale bars: 10 μm (A); 1 μm (B and C). Hair cell ages: B–E: P2 plus 3–4 days in vitro, F and G: P2–3 plus 2–4 days in vitro.

Figure 1. Auditory mechanotransduction phenotype of young postnatal Myo15^sh2/sh2 mice

A and B, SEM images of Myo15^sh2/sh2 OHCs at postnatal day 9 (P9) (A) and 19 (B). Insets show magnified images of stereocilia. Note stereocilia links in A and their absence in B. C, auditory brainstem responses in P16 homozygous (left) and heterozygous (right) Myo15^sh2 mice. Stimulus type and intensity (dB SPL, sound pressure level) are indicated. Arrows mark the stimulus onset. D, epifluorescent images of Myo15^sh2/sh2 organ of Corti explants (harvested at P3 and kept 3 days in vitro) after incubations with 5 μm FM1-43 for 60 s (left), FM1-43 and 1 mm GdCl₃ for 60 s (middle), or FM3-25 for 6 min (right). E, time course of FM1-43 uptake in Myo15^sh2/sh2 hair cells measured at the confocal optical plane below the cuticular plate. Insets show the actual fluorescent images before and immediately after application of 5 μm of the dye to the apical surface of OHCs. Scale bars: 1 μm (A, B); 10 μm (D). All cells (A and B, D and E) were located approximately at the end of the first (apical) cochlear turn.

Figure 3. Properties of mechano-electrical transduction in Myo15^sh2/sh2 OHCs

A, voltage–current relationship of the maximal mechanotransduction current evoked by ramp deflections of the bundle at different holding potentials (inset). B, mechanotransduction currents recorded at −90 mV and +90 mV holding potentials in the same cell. C, mechanotransduction current (left) evoked by a brief bundle deflection before (○) and after (▪) a longer step-like deflection of stereocilia by 0.6 μm at −70 mV holding potential. Displacement–current relationships (right) were fitted to a first order Boltzmann function to determine a shift due to adaptation (0.5 μm, indicated by arrow). Access resistances (MΩ): A −40.6; B −37.8; C −74.