Mutations in myosin-VIIa (MYO7A) cause Usher syndrome type 1, characterized by combined deafness and blindness. MYO7A is proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex, but conclusive evidence is lacking. Here we report that multiple MYO7A isoforms are expressed in the mouse cochlea. In mice with a specific deletion of the canonical isoform (Myo7a-ΔC mouse), MYO7A is severely diminished in inner hair cells (IHCs), while expression in outer hair cells is affected tonotopically. IHCs of Myo7a-ΔC mice undergo normal development, but exhibit reduced resting open probability and slowed onset of MET currents, consistent with MYO7A's proposed role in tensioning the tip link. Mature IHCs of Myo7a-ΔC mice degenerate over time, giving rise to progressive hearing loss. Taken together, our study reveals an unexpected isoform diversity of MYO7A expression in the cochlea and highlights MYO7A's essential role in tensioning the hair cell MET complex.

Sound detection in auditory sensory hair cells depends on the deflection of the stereocilia hair bundle which opens mechano-electric transduction (MET) channels. Adaptation is hypothesized to be a critical property of MET that contributes to the auditory system's wide dynamic range and sharp frequency selectivity. Our recent work using a stiff probe to displace hair bundles showed that the fastest adaptation mechanism (fast adaptation) does not require calcium entry. Using fluid-jet stimuli, others obtained data showing only a calcium-dependent fast adaptation response. Because cochlear stereocilia do not move coherently and the hair cell response depends critically on the magnitude and time course of the hair bundle deflection, we developed a high-speed imaging technique to quantify this deflection in rat cochlear hair cells. The fluid jet delivers a force stimulus, and force steps lead to a complex time course of hair bundle displacement (mechanical creep), which affects the hair cell's macroscopic MET current response by masking the time course of the fast adaptation response. Modifying the fluid-jet stimulus to generate a hair bundle displacement step produced rapidly adapting currents that did not depend on membrane potential, confirming that fast adaptation does not depend on calcium entry. MET current responses differ with stimulus modality and will shape receptor potentials of different hair cell types based on their in vivo stimulus mode. These transformations will directly affect how stimuli are encoded.

Hair cells detect sound and motion through a mechano-electric transduction (MET) process mediated by tip links connecting shorter stereocilia to adjacent taller stereocilia. Adaptation is a key feature of MET that regulates a cell’s dynamic range and frequency selectivity. A decades-old hypothesis proposes that slow adaptation requires myosin motors to modulate the tip-link position on taller stereocilia. This “motor model” depended on data suggesting that the receptor current decay had a time course similar to that of hair-bundle creep (a continued movement in the direction of a step-like force stimulus). Using cochlear and vestibular hair cells of mice, rats, and gerbils, we assessed how modulating adaptation affected hair-bundle creep. Our results are consistent with slow adaptation requiring myosin motors. However, the hair-bundle creep and slow adaptation were uncorrelated, challenging a critical piece of evidence upholding the motor model. Considering these data, we propose a revised model of hair cell adaptation.

Hair cells of the auditory and vestibular systems transform mechanical input into electrical potentials through the mechanoelectrical transduction process (MET). Deflection of the mechanosensory hair bundle increases tension in the gating springs that open MET channels. Regulation of MET channel sensitivity contributes to the auditory system’s precision, wide dynamic range and, potentially, protection from overexcitation. Modulating the stiffness of the gating spring modulates the sensitivity of the MET process. Here, we investigated the role of cyclic adenosine monophosphate (cAMP) in rat outer hair cell MET and found that cAMP up-regulation lowers the sensitivity of the channel in a manner consistent with decreasing gating spring stiffness. Direct measurements of the mechanical properties of the hair bundle confirmed a decrease in gating spring stiffness with cAMP up-regulation. In parallel, we found that prolonged depolarization mirrored the effects of cAMP. Finally, a limited number of experiments implicate that cAMP activates the exchange protein directly activated by cAMP to mediate the changes in MET sensitivity. These results reveal that cAMP signaling modulates gating spring stiffness to affect auditory sensitivity.

37Sound detection in auditory sensory hair cells depends on the deflection of the 38 stereocilia hair bundle, which opens mechano-electric transduction (MET) channels. 39 Adaptation is hypothesized to be a critical property of MET that contributes to the wide 40 dynamic range and sharp frequency selectivity of the auditory system. Historically, 41 adaptation was hypothesized to have multiple mechanisms, all of which require calcium 42 entry through MET channels. Our recent work using a stiff probe to displace hair bundles 43 showed that the fastest adaptation mechanism (fast adaptation) does not require 44 calcium entry. Using a fluid-jet stimulus, others obtained data showing only a calcium-45 dependent fast adaptation response. Here, we identified the source of this discrepancy. 46Because the hair cell response to a hair bundle stimulus depends critically on the 47 magnitude and time course of the hair bundle deflection, we developed a high-speed 48 imaging technique to quantify this deflection. The fluid jet delivers a force stimulus, and 49 step-like force stimuli lead to a complex time course of hair bundle displacement 50 (mechanical creep), which affects the hair cell's macroscopic MET current response by 51 masking the time course of the fast adaptation response. Modifying the fluid-jet stimulus 52to generate a step-like hair bundle displacement produced rapidly adapting currents that 53 did not depend on membrane potential. This indicated that fast adaptation does not 54 depend on calcium entry. We also confirmed the presence of a calcium-dependent slow 55 adaptation process. These results confirm the existence of multiple adaptation 56 processes: a fast adaptation that is not driven by calcium entry and a slower calcium-57 dependent process. 58 Significance Statement: 59 3Mechanotransduction by sensory hair cells represents a key first step for the sound 60 sensing ability in vertebrates. The sharp frequency tuning and wide dynamic range of 61 sound sensation are hypothesized to require a mechanotransduction adaptation 62 mechanism. For decades, it had been accepted that all adaptation mechanisms require 63 calcium entry into hair cells. However, more recent work indicated that the apparent 64 calcium dependence of the fastest adaptation differs with the method of cochlear hair 65 cell stimulation. Here, we reconcile existing data and show that calcium entry does not 66 drive the fastest adaptation process, independent of the stimulation method. 67 Introduction: 68 Inner ear hair bundles comprise an array of graded length stereocilia that are 69 organized in a staircase manner. Positive hair bundle deflection increases tension in a 70 filamentous tip link that connects adjacent rows of stereocilia (1-3). Changes in tip-link 71 tension result in the gating of mechano-electric transduction (MET) ion channels. 72 MET adaptation is hypothesized to extend the dynamic range of the cell, control 73 the channel's operating point, and filter incoming stimuli (4-6). Adaptation is identified 74 by two phenom...