Modulation of outer hair cell electromotility by cochlear supporting cells and gap junctions

Abstract

Outer hair cell (OHC) or prestin-based electromotility is an active cochlear amplifier in the mammalian inner ear that can increase hearing sensitivity and frequency selectivity. In situ, Deiters supporting cells are well-coupled by gap junctions and constrain OHCs standing on the basilar membrane. Here, we report that both electrical and mechanical stimulations in Deiters cells (DCs) can modulate OHC electromotility. There was no direct electrical conductance between the DCs and the OHCs. However, depolarization in DCs reduced OHC electromotility associated nonlinear capacitance (NLC) and distortion products. Increase in the turgor pressure of DCs also shifted OHC NLC to the negative voltage direction. Destruction of the cytoskeleton in DCs or dissociation of the mechanical-coupling between DCs and OHCs abolished these effects, indicating the modulation through the cytoskeleton activation and DC-OHC mechanical coupling rather than via electric field potentials. We also found that changes in gap junctional coupling between DCs induced large membrane potential and current changes in the DCs and shifted OHC NLC. Uncoupling of gap junctions between DCs shifted NLC to the negative direction. These data indicate that DCs not only provide a physical scaffold to support OHCs but also can directly modulate OHC electromotility through the DC-OHC mechanical coupling. Our findings reveal a new mechanism of cochlear supporting cells and gap junctional coupling to modulate OHC electromotility and eventually hearing sensitivity in the inner ear.

Figure 1. Modulation of Deiters cell (DC) membrane potential and current on outer hair cell (OHC) electromotility.

A: A micrograph of double patch clamp recording between the DC and OHC in a DC-OHC pair. B: Double patch clamp recording between the DC and the OHC. There is neither transjunctional current nor electric conductance between the DC and the OHC. C: Changes in the holding potential of the DC alter OHC electromotility associated nonlinear capacitance (NLC). Both DC and OHC were recorded under voltage clamp. The bottom traces represent the different holding potentials at the DC. D: Changes in the holding current of the DC alter OHC NLC. The Deiters cell was clamped at different currents under the current clamp.

Figure 2. Breaking of mechanical connection between DC and OHC abolishes the effect of DC membrane potential on OHC electromotility.

A: A micrograph of double patch clamp recording in a pair of DC-OHC. An arrow indicates dissociation of mechanical connection between the DC and the basal pole of the OHC. B: Dissociation abolishes the effect of DC membrane potential on OHC electromotility.

Figure 3. The effect of DC membrane potential on the OHC frequency responses and distortion products.

A: The frequency spectrum and distortion products of OHC response to a two-sinusoidal electrical stimulation (f₁ = 976.56 Hz, f₂ = 1171.87 Hz, and V_p−p = 25 mV) in patch-clamp recording. Inset: A waveform of OHC response. B: The amplitudes of OHC frequency responses at different DC holding potentials. The amplitudes are normalized to those at DC holding potential of −120 mV. C: OHC distortion products at different holding potentials of the DCs. Depolarization of the DCs decreased OHC distortion products. Asterisks indicate p<0.05 (t test). D: Membrane potentials of DCs alter the OHC distortion product. The sum frequency distortion (f₁+f₂) was displayed. Perfusion of 1 mM LuCl₃ reduced the effect.

Figure 4. The effect of gap junctional coupling between DCs on OHC electromotility.

A: A micrograph of a DC-OHC pair. An OHC is connected with two DCs. B: Membrane potential and current changes in DCs by uncoupling of gap junctions. Uncoupling reduced DC's membrane current and shifted zero-current potential to negative. C: Influence of gap junctional coupling between DCs on OHC NLC. The black and red lines represent the OHC membrane capacitance measured at DCs coupling and uncoupling, respectively, which was achieved by mechanical breaking. Uncoupling of gap junctions between DCs shifted the OHC NLC curve to negative. The V_pk was −21.8 and −40.3 mV and the NLC was 22.5 and 23.6 pF for DC coupling and uncoupling, respectively. D–E: Uncoupling of gap junctions between DCs induced changes in OHC NLC and V_pk-shift.

Figure 5. Destruction of DC cytoskeleton eliminates the effect of electrical stimulations in the DCs on OHC electromotility.

Trypsin (0.25%) was only filled into the DC patch pipette. A: The DC holding potential can alter NLC at the beginning of whole-cell recording. B: The OHC NLC was recorded at 15 min after formation of whole-cell recording. The effect of DC membrane potential on NLC disappeared.

Figure 6. The effect of turgor pressure changes in the DC on OHC electromotility in a DC-OHC pair.

The peak voltage (V_pk) of NLC in the OHC was continuously recorded by use of a phase-tacking technique (Panel A). The DC was voltage-clamped at −40 mV under the whole-cell configuration and its turgor pressure was altered through the patch pipette (Panel B). Increase of turgor pressure in the DC caused the V_pk of OHC NLC shifted to negative voltage.