Rat Auditory Inner Hair Cell Mechanotransduction and Stereociliary Membrane Diffusivity Are Similarly Modulated by Calcium

Abstract

Summary The lipid bilayer plays a pivotal role in force transmission to many mechanically-gated channels. We developed the technology to monitor membrane diffusivity in order to test the hypothesis positing that Ca 2+ regulates open probability ( P o ) of cochlear hair cell mechanotransduction (MET) channels via the plasma membrane. The stereociliary membrane was more diffusive (9x) than the basolateral membrane. Elevating intracellular Ca 2+ buffering or lowering extracellular Ca 2+ reduced stereociliary diffusivity and increased MET P o . In contrast, prolonged depolarization increased stereociliary diffusivity and reduced MET P o . No comparable effects were noted for soma measurements. Although MET channels are located in the shorter stereocilia rows, both rows had similar baseline diffusivity and showed similar responses to Ca 2+ manipulations and MET channel blocks, suggesting that diffusivity is independent of MET. Together, these data suggest that the stereociliary membrane is a component of a calcium-modulated viscoelastic-like element regulating hair cell mechanotransduction.

A two-photon FRAP protocol to measure the stereociliary membrane diffusivity in rat cochlear hair cells

“… Analyze the FRAP data to generate FRAP curve using Image J. Irreversible bleaching of di-3-ANEPPDHQ should show no recovery in the fluorescence signal after photobleaching (see Figures S1C and S1D in George et al. (2020) ).…”

“…In this section, we describe the important parameters needed for an accurate estimation of the diffusion coefficient D from the FRAP curve extracted from the IHC stereocilia. For the detailed description of how to calculate the diffusion coefficient D using Mathematic a, please refer to George et al. (2020) and the code associated to the implementation of the model is available through Mendeley Data [Mendeley Data: https://doi.org/10.17632/3wrd9xp4gc.1 ].…”

“…The excitation photoselection effect can be corrected by rotating the laser polarization with a λ/2 (half-wave) plate, thus converting filled stereocilia to unfilled stereocilia ( Figure 7 ) ( George et al., 2020 ).

Figure 7 Example of excitation photoselection effect

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“…We also detail two-photon FRAP microscope setup and calibration, as well as FRAP parameter setting and data analysis. For complete details on the use and execution of this protocol, please refer to George et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to George et al. (2020) .…”