Electrokinetic properties of the mammalian tectorial membrane

Abstract

The tectorial membrane (TM) clearly plays a mechanical role in stimulating cochlear sensory receptors, but the presence of fixed charge in TM constituents suggests that electromechanical properties also may be important. Here, we measure the fixed charge density of the TM and show that this density of fixed charge is sufficient to affect mechanical properties and to generate electrokinetic motions. In particular, alternating currents applied to the middle and marginal zones of isolated TM segments evoke motions at audio frequencies (1-1,000 Hz). Electrically evoked motions are nanometer scaled (∼5-900 nm), decrease with increasing stimulus frequency, and scale linearly over a broad range of electric field amplitudes (0.05-20 kV/m). These findings show that the mammalian TM is highly charged and suggest the importance of a unique TM electrokinetic mechanism.

Fig. 1.

Electromechanical properties of the TM. Schematic drawing of the TM showing negative fixed-charge constituents attached to a network of mechanical springs (collagen fibers). In response to external pressure, the negative fixed charge and collagen fibers resist compression through electrostatic repulsion effects and mechanical forces, respectively.

Fig. 2.

Fixed-charge density of the TM. (A) Schematic drawings of the microaperture setup showing side views of an isolated TM positioned over a circular microaperture. (Left) The microaperture creates a fluid path from the overlying bath of AE to the underlying microfluidics channel (test bath) perfused with AE-like solutions with variable KCl concentrations. (Right) The TM acts as an electrochemical barrier between the overlying bath and underlying fluid channel. The potential difference between the baths (V_D = V_T − V_B) was recorded with Ag/AgCl electrodes that were placed in contact with the two baths. (B) Schematic drawing of the microaperture setup showing the top view of the TM positioned over the microaperture in the region near Hensen’s stripe and the marginal zone. (Inset) TM segment overlying the microaperture using 40× magnification. (C) Voltage was plotted as a function of test bath KCl concentration. Best-fit estimates to the median voltages yielded c_f (–7.1 ± 2.0 mmol/L; n = 5 TM preparations). Vertical lines and boxes denote extreme values and the extent of the interquartile range, respectively. Horizontal lines through the boxes denote the median values. Reducing the bath pH from 7.3 to 3.5 caused voltage measurements to change polarity and decrease in magnitude (3.0 mmol/L).

Fig. 3.

TM electrokinetic response. (A) The microaperture setup was used to deliver electric fields to the TM with a pair of Ag/AgCl-stimulating electrodes. Electrically evoked displacements were measured using computer microvision and DOCM systems (Materials and Methods). Voltages were delivered with Ag/AgCl electrodes positioned in the top bath and in a reservoir connecting to the underlying fluid channel. Electric fields were computed based on the geometry of the microaperture, and electrical current was measured across a resistor that was placed in series with the microaperture chamber during voltage application. (B) Transverse displacements were sinusoidal and largest in the regions of the TM directly overlying the microaperture and decreased with radial distance away from the microaperture. A typical TM segment excised from the middle turn exhibited electrically evoked displacements up to ∼45 nm in response to electrical stimuli applied in the middle zone region directly overlying the microaperture (10 Hz; 8 kV/m). Motion amplitudes varied depending on whether the TM sample was a basal or an apical segment. (C) Displacements scaled linearly with electric field magnitude for TM samples excised from the middle turn of the cochlea (n = 4 TM preparations). (D) (Upper) Displacement amplitudes decreased with increasing stimulus frequency (40–1,000 Hz) with a slope of –1 (solid line) for a typical apical TM segment. Black dots denote the mean value of TM displacement. (Lower) Phase angle of TM displacement as a function of frequency. Vertical lines with horizontal bars denote SEM.

Fig. 4.

TM electrokinetics near hair cell ion channels. (A) Schematic drawing of a cochlear partition showing the orientation of the TM relative to OHC stereocilia and tip links. (B) Inset shows the OHC ion channel as a point source with radial electric fields (blue arrows) acting on TM fixed-charge macromolecules locally over small distances r. (C) Model predictions of electric field magnitudes as a function of radial distance r from the opening of the ion channel. Electric fields were estimated based on experimental values for single-channel transduction currents, resistivity of the ionic environment, and the radius of the ion channel from previous data (–55). The shaded region labeled “This study” denotes the range of electric field (10³–10⁴ V/m) magnitudes applied in the microaperture chamber with values corresponding to ∼7–30 nm from the ion channel opening. The shaded region labeled “Ref. ” denotes electric fields (∼1 V/m) measured at ∼1 µm from the tips of hair bundles in the bullfrog’s sacculus (51). In both cases, radial distances r are significantly larger than the TM’s space charge layer thickness (34).