A large-conductance calcium-selective mechanotransducer channel in mammalian cochlear hair cells

Abstract

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mM calcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.

Figure 1.

Method for mechanically stimulating inner hair cell bundles. A view of the rat organ of Corti from the apical region showing hair bundles of three IHCs and four row-one OHCs is shown. The IHC can be deflected with a fire-polished glass probe ∼6 μm in diameter that fits into the broad U-shaped hair bundle. The other end of the probe is attached to a piezoelectric stack actuator. For the outer hair cells, a smaller probe, ∼3 μm diameter, is adequate. Note that at least three rows of stereocilia can be seen on the IHC bundles. The postnatal age of the rat was P8.

Figure 2.

Comparison of MT currents from inner and outer hair cells. A, Responses of an OHC in the apical turn tuned to ∼4 kHz. B, Responses of an IHC, also from the apical turn. C, Responses of an IHC in the middle turn tuned to ∼14 kHz. Each panel shows average currents in response to hair bundle deflections, positive steps for motion away from the modiolus. Note that adaptation is faster in the apical OHC than in the apical IHC. The relationship between peak MT current, I, and bundle displacement, X, is shown at the bottom. Smooth curves are as follows. A, I = I_max/[1 + exp(z₀ × (X₀ − X)], where I_max = 0.75 nA, z₀ = 11.3 μm⁻¹, and X₀ = 0.24 μm. B, C, I = I_max/[1 + {exp(z₀ × (X₀ − X))}{1 + exp(z₁ × (X₁ − X))}], where (B) I_max = 0.98 nA, z₀ = 6.5 μm⁻¹, z₁ = 13 μm⁻¹, and X₀ = X₁ = 0.14 μm, and (C) I_max = 0.76 nA, z₀ = 5 μm⁻¹, z₁ = 20 μm⁻¹, and X₀ = X₁ = 0.13 μm. Postnatal ages were P7 (A), P8 (B), and P8 (C).

Figure 3.

Single MT channels in an apical-turn inner hair cell. A, Examples of unitary responses to a hair bundle displacement showing closed (C) and open (O) states of the channel. The time course of the 0.6 μm displacement step is shown at the top. Examples were chosen to illustrate full openings. B, Average response to 10 stimuli. Peak current was 12 pA. C, Amplitude histogram of responses fitted with a pair of Gaussians indicates a single-channel current of 16 pA, with a holding potential of −84 mV. The postnatal age of the rat was P10.

Figure 4.

Single MT channels in a middle-turn inner hair cell. A, Unitary responses showing closed (C) and open (O) states of the channel for different hair bundle displacements. The amplitude histogram for the two largest stimuli is shown below, indicating a single-channel current of 16 pA, with a holding potential of −84 mV. B, Ensemble averages for different hair bundle displacements. Note the fast adaptation in the small responses and the additional activity after the step for the largest. C, Probability of channel opening versus displacement (X) for this channel (circles) and for another channel from an apical inner hair cell (triangles). Smooth curve: P_open = 1/(1 + exp(z₀ × (X₀ − X))), where X₀ = 0.27 μm, and z₀ = 10 μm⁻¹. The postnatal age of the rat was P10.

Figure 5.

Single MT channels in a two outer hair cells. A, Apical turn. B, Middle turn. In each case, unitary responses are shown for displacement steps of 0.6 μm in A and 0.4 μm in B. Below the single-channel records are the ensemble average current and the amplitude histogram indicating a single-channel current of 8.3 pA in A and 11.0 pA in B. Holding potential of −84 mV. The postnatal ages of the rats were P11 (A) and P8 (B).

Figure 6.

Scanning electron micrographs of hair bundles in the apical turn. A, Inner hair cell. B, First-row outer hair cell. Scale bars, 2.0 μm. These are representative examples of hair bundles from a P15 rat used for stereociliary and tip link counts.

Figure 7.

Effects of calcium on the MT currents of apical inner hair cells. A, Average MT currents in response to step deflections of the hair bundle recorded during superfusion with saline containing 1.5 and 0.02 mm calcium. Reducing extracellular calcium increased the maximum current amplitude and slowed the rate of adaptation. The holding potential is −84 mV. B, Current–voltage relationships in another apical inner hair cell before and after lowering the extracellular calcium from 1.5 to 0.02 mm. The postnatal ages of rats were P7 (A) and P8 (B).

Figure 8.

Reversal potential of inner hair cell MT current in normal and high calcium. A, MT currents for saturating hair bundle deflections occurring during a voltage ramp: normal (1.5 mm) calcium saline (top), high (100 mm) calcium with no other permeant ion (middle), and bundle stimuli (bottom). B, Current–voltage relationships for the MT channel in normal calcium (filled circles) and high calcium (crosses). The reversal potential was +5 mV in normal saline and +31 mV in 100 mm calcium. The postnatal age of the rat was P11.

Figure 9.

A model for differences in MT channel structure to account for variation in conductance and calcium permeability. A, Hypothetical structure of the MT channel protein in a low conductance and high conductance channel. Negatively charged residues in a putative outer vestibule would locally concentrate the ions and increase channel conductance as found in BK channels (Brelidze et al., 2003). These residues may be absent on the low conductance version. The existence of a wide outer vestibule in the MT channel was suggested (Farris et al., 2004) on the basis of open channel block at the external surface by large molecules such as curare. B, Effects of reducing the external sodium concentration and ionic strength on MT currents in an apical IHC (top) and an apical OHC (bottom) that should differ twofold in their channel conductance. The fractional reduction in maximum current is larger in the OHC.