The Frequency Response of Outer Hair Cell Voltage-Dependent Motility Is Limited by Kinetics of Prestin

Abstract

The voltage-dependent protein SLC26a5 (prestin) underlies outer hair cell electromotility (eM), which is responsible for cochlear amplification in mammals. The electrical signature of eM is a bell-shaped nonlinear capacitance (NLC), deriving from prestin sensor-charge (Q) movements, which peaks at the membrane voltage, V, where charge is distributed equally on either side of the membrane. Voltage dependencies of NLC and eM differ depending on interrogation frequency and intracellular chloride, revealing slow intermediate conformational transitions between anion binding and voltage-driven Q movements. Consequently, NLC exhibits low-pass characteristics, substantially below prevailing estimates of eM frequency response. Here we study in guinea pig and mouse of either sex synchronous prestin electrical (NLC, Q) and mechanical (eM) activity across frequencies under voltage clamp (whole cell and microchamber). We find that eM and Q magnitude and phase correspond, indicating tight piezoelectric coupling. Electromechanical measures (both NLC and eM) show dual-Lorentzian, low-pass behavior, with a limiting (τ) time constant at V of 32.6 and 24.8 μs, respectively. As expected for voltage-dependent kinetics, voltage excitation away from V has a faster, flatter frequency response, with our fastest measured τ for eM of 18.2 μs. Previous observations of ultrafast eM (τ ≈ 2 μs) were obtained at offsets far removed from V We hypothesize that trade-offs in eM gain-bandwith arising from voltage excitation at membrane potentials offset from V influence the effectiveness of cochlear amplification across frequencies. Of two types of hair cells within the organ of Corti, inner hair cells and outer hair cells, the latter evolved to boost sensitivity to sounds. Damage results in hearing loss of 40-60 dB, revealing amplification gains of 100-1000× that arise from voltage-dependent mechanical responses [electromotility (eM)]. eM, driven by the membrane protein prestin, may work beyond 70 kHz. However, this speed exceeds, by over an order of magnitude, kinetics of typical voltage-dependent membrane proteins. We find eM is actually low pass in nature, indicating that prestin bears kinetics typical of other membrane proteins. These observations highlight potential difficulties in providing sufficient amplification beyond a cutoff frequency near 20 kHz. Nevertheless, observed trade-offs in eM gain-bandwith may sustain cochlear amplification across frequency.

Outer hair cell electromotility is low-pass filtered relative to the molecular conformational changes that produce nonlinear capacitance

“…However, these data were obtained at voltage offsets far removed from V h , which has questionable physiological significance. Based on our measures (Santos-Sacchi and Tan, 2018), we estimate that their depolarized offset from V h would have been ∼65 mV. Indeed, by exploring at an offset potential near V h , we recently demonstrated that eM measured with the microchamber displays significant low-pass behavior (Santos-Sacchi and Tan, 2018).…”

“…As noted above, whole-cell NLC determination was made following stray capacitance removal (Santos-Sacchi et al, 1998; Santos-Sacchi and Song, 2016; Santos-Sacchi, 2018; Santos-Sacchi and Tan, 2018). Patch pipettes were coated with M-coat (Micro Measurements) to reduce stray capacitance.…”

“…The patch electrode provided a fixed point near the basal end of the cell. Since movements were measured at the apical pole of the cell and the cell was held physically at the point of electrode insertion, estimates of full whole-cell movements were obtained from the ratio of apical/basal partitioning at the point of electrode insertion as in microchamber experiments (Frank et al, 1999; Santos-Sacchi and Tan, 2018). Resultant movements were analyzed by FFT in MATLAB.…”

“…In the model, we are able to directly measure imposed membrane voltage so we can compare results from these approaches to actual values. The generated charge in the model is taken as the equivalent of eM, since we have shown that OHC eM and prestin charge movement are directly coupled (Santos-Sacchi and Tan, 2018). The model we use to confirm our voltage corrections has been fully described before (Santos-Sacchi and Song, 2014a), and includes a slow, stretched exponential intermediary transition between chloride binding transitions and voltage-dependent charge transitions, the latter corresponding to eM.…”

Timing of the reticular lamina and basilar membrane vibration in living gerbil cochleae

“…Although delays for prestin-associated currents in vitro (Santos-Sacchi and Tan, 2018) are longer than the latency difference between the reticular lamina and basilar membrane vibration in vivo, they vary with the membrane voltage of outer hair cells. Voltage excitation away from the resting membrane potential has been shown to have a faster response.…”