The effects of Tmc1 Beethoven mutation on mechanotransducer channel function in cochlear hair cells

Abstract

Sound stimuli are converted into electrical signals via gating of mechano-electrical transducer (MT) channels in the hair cell stereociliary bundle. The molecular composition of the MT channel is still not fully established, although transmembrane channel-like protein isoform 1 (TMC1) may be one component. We found that in outer hair cells of Beethoven mice containing a M412K point mutation in TMC1, MT channels had a similar unitary conductance to that of wild-type channels but a reduced selectivity for Ca(2+). The Ca(2+)-dependent adaptation that adjusts the operating range of the channel was also impaired in Beethoven mutants, with reduced shifts in the relationship between MT current and hair bundle displacement for adapting steps or after lowering extracellular Ca(2+); these effects may be attributed to the channel's reduced Ca(2+) permeability. Moreover, the density of stereociliary CaATPase pumps for Ca(2+) extrusion was decreased in the mutant. The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca(2+). Consistent with this idea, the adaptive shift in the current-displacement relationship when hair bundles were bathed in endolymph-like Ca(2+) saline was usually abolished by raising the intracellular Ca(2+) concentration.

Figure 1.

MT channel conductance and Ca²⁺ selectivity in Tmc1^Bth/Bth mice. (A) Four examples of single MT channel currents, recorded in a P5 apical IHC of Tmc1^+/+ mice in response to 0.1-µm step displacements of the hair bundle; below is the ensemble average of 14 presentations. (B) Amplitude histogram showing single-channel current of 6.2 pA. (C) Four examples of single MT channel currents recorded in a P5 apical IHC of Tmc1^Bth/Bth mice in response to 0.1-µm step displacements of the hair bundle; below is the ensemble average of 12 presentations. (D) Amplitude histogram showing single-channel current of 6.3 pA; both B and D were at a −84-mV holding potential in 1.5 mM of external Ca²⁺. (E) Bar plot showing collected single-channel currents in IHCs, apical OHCs, and basal OHCs for Tmc1^+/+ and Tmc1^Bth/Bth. Number of cells tested is shown above the columns. (F) Protocol for determining Ca²⁺ selectivity: mechanical hair bundle stimulus (top) evoking MT current (bottom) during voltage ramp from −40 to 70 mV. (G) Examples of MT current–voltage relationships recorded in apical OHCs from Tmc1^+/+ and Tmc1^Bth/Bth mice in the voltage region around reversal potential. (H) Collected reversal potentials (left ordinate) and P_Ca/PCs (right ordinate) for Tmc1^+/+ and Tmc1^Bth/Bth in Tmc2^+/+ and Tmc2^−/− backgrounds. Numbers of OHCs tested are shown above the columns. Apical OHCs: P4–P6 mice. Error bars represent means ± SD.

Figure 2.

Effects of low endolymph-like Ca²⁺ on MT current in OHCs of Tmc1^Bth/Bth. (A) MT currents recorded from a P5 OHC of Tmc1^+/+ mouse at −84 mV when the bundle was bathed in saline with 1.5 mM, 0.04 mM, and return control 1.5 mM of extracellular Ca²⁺. Note that in 0.04 mM Ca²⁺, the resting open probability increased in Tmc1^+/+. (B) Current–displacement relations in Tmc1^+/+, peak, I (top), and normalized (I/I_max; bottom) MT currents recorded from the OHC as a function of bundle displacement. (C) MT currents in P5 OHCs of Tmc1^Bth/Bth mice at −84 mV when the bundle was bathed in saline with 1.5 mM, 0.04 mM, and return control 1.5 mM of extracellular Ca²⁺. (D) Current–displacement relations in Tmc1^Bth/Bth and maximum and normalized MT currents recorded from an OHC as a function of bundle displacement. The plots demonstrate a leftward shift of the MT current–displacement relation in Tmc1^+/+ (B) but not in Tmc1^Bth/Bth mice (D). Sets of points fitted with Eq. 1 with the following parameters: B, I_MAX = 1.2 nA, X_0.5 = 38 nm, X_S = 15 nm, and 1.5 mM Ca²⁺; I_MAX = 1.7 nA, X_0.5 = 5 nm, X_S = 15 nm, and 0.04 mM Ca²⁺. D, I_MAX = 1.14 nA, X_0.5 = 46 nm, X_S = 16 nm, and 1.5 mM Ca²⁺; I_MAX = 1.68 nA, X_0.5 = 41 nm, X_S = 19 nm, and 0.04 mM Ca²⁺. In both genotypes, the maximum current was increased in 0.04 mM Ca²⁺ relative to 1.5 mM Ca²⁺. In B and D, hair bundle displacement was calculated from the piezoelectric driving voltage (V_piezo) using the calibrations described in Results.

Figure 3.

Adaptive shift in a paired-pulse protocol is reduced in OHCs of Tmc1^Bth/Bth. (A and B) Superimposed MT currents recorded from a P4 OHC in response to paired-pulse hair bundle stimulation, with the second pulse preceded by a 10-ms adapting step. Holding potential (HP) was −84 mV (A) and +96 mV (B). (C–E) Current–displacement relationships for first pulse (S1; closed squares) and second pulse (S2; open squares) at −84, +96, and return to −84 mV. Change in holding potential produced by a 100-ms depolarizing step starting 8 ms before the mechanical stimuli. MT currents, I, scaled to maximum current, I_MAX; displacements were determined from piezoelectric driver voltage (V_piezo) by calibration as described in Results. Note that the adaptive shift in the current–displacement relationship at −84 mV was abolished at +96 mV. Each set of points fit with Eq. 1 with the following parameters: C, I_MAX = 1.4 nA, X_0.5 = 42 nm, and X_S = 12 nm, S1; I_MAX = 1.38 nA, X_0.5 = 60 nm, and X_S = 12 nm, S2; D, I_MAX = 1.2 nA, X_0.5 = 40 nm, and X_S = 18 nm, S1; I_MAX = 1.2 nA, X_0.5 = 39 nm, and X_S = 15 nm, S2; E, I_MAX = 1.4 nA, X_0.5 = 48 nm, and X_S = 12 nm, S1; I_MAX = 1.39 nA, X_0.5 = 70 nm, and X_S = 15 nm, S2. Recordings from apical P4 OHCs of Tmc1^+/+; similar results were seen in six OHCs of P4–P6 mice. (F) Paired-pulse experiment in which the amplitude of the adapting step (A) was varied, shown for one test pulse. (G) For each adapting step, a family of test pulses was presented to determine the current–displacement relationships, shown for control (closed squares) and three adapting steps. For each adapting step, the current–displacement relationship was fitted with a single Boltzmann to determine ΔX, the shift in half-amplitude. (H) The shift, ΔX, is plotted against (A), the size of the adapting step for five OHCs of Tmc1^+/+ (closed circles) and five OHCs of Tmc1^Bth/Bth (crosses). All points are for each genotype fitted with a straight line: slope, 0.52 ± 0.02 in Tmc1^+/+ and 0.33 ± 0.01 in Tmc1^Bth/Bth; apical OHCs of P4–P6 mice.

Figure 4.

Adaptive shift in a paired-pulse protocol in 2.5 mM of intracellular Ca²⁺. (A and B) Superimposed MT currents recorded from a P4 OHC in response to paired-pulse hair bundle stimulation, with the second pulse preceded by an adapting step as in Fig. 3. Holding potential (HP) was −84 (A) and +96 mV (B). (C–E) Current–displacement relationships for first pulse (S1; closed circles) and second pulse (S2; crosses) recorded at −84, +96, and return to −84 mV. MT currents, I, scaled to maximum current I_MAX; displacements were determined from piezoelectric driver voltage (V_piezo) by calibration as described in Results. Note that the adaptive shift in the current–displacement relationship persisted at −84 mV, even though the recording pipette contained 2.5 mM Ca²⁺, and this shift was abolished at +96 mV. Sets of points fit with the Boltzmann equation (see Materials and methods).

Figure 5.

Adaptive shift in different extracellular and cytoplasmic Ca²⁺ concentrations. (A) Superimposed MT currents in paired-pulse protocol for 1.5 mM of external Ca²⁺ (Ca_O) and 2.5 mM of internal Ca²⁺ (Ca_i). (B) Current–displacement relations for first pulse (control; closed squares) and second pulse (+step; open squares) of records in A. (C) Superimposed MT currents in paired-pulse protocol for 0.04 mM Ca_O and 0 Ca_i buffered with 1 mM EGTA. (D) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in C. (E) Superimposed MT currents in paired-pulse protocol for 0.04 mM Ca_O and 2.5 mM Ca_i. (F) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in E. Note that there were adaptive shifts in B and D, but not in F. (G) Current–voltage relationships of MT channel as in Fig. 1 G, with 1.5 mM Ca_O and 2.5 mM Ca_i (closed circles) and 0.04 mM Ca_O and 2.5 mM Ca_i (open circles). (H) Reversal potentials and permeability ratios, P_Ca/PCs, with initial exposure to Ca_o = 1.5 mM (closed circles) and prolonged prior exposure to Cao = 0.04 mM (open circles) as a function of intracellular Ca²⁺ concentration, Ca_i. Error bars represent the mean ± SD, with the number of experiments given above the points. Theoretical values are calculated from Eq. 1, assuming Ca_i = 0 mM (crosses). All recordings were in apical OHCs of P4–P5 mice.

Figure 6.

Hair bundle expression of PMCA2 Ca²⁺ pump is reduced in Beethoven mice. (A) Immunofluorescent labeling of the apical coil cochlea from a Tmc1^+/+ P6 mouse. (Top) Phalloidin-labeled hair bundles of three rows of OHCs above and one row of IHCs below. (Middle) PMCA2 labeling in which OHC bundles label more than IHCs. (Bottom) A merged image. (B) Immunofluorescent labeling of an apical cochlear coil from a Tmc1^Bth/Bth P6 mouse. (Top) Phalloidin-labeled hair bundles of three rows of OHCs above and one row of IHCs below. (Middle) PMCA2 labeling in which OHC bundles label more than IHCs. (Bottom) A merged image. Label in Tmc1^Bth/Bth is much less pronounced than that in Tmc1^+/+ mice. (C) Collected measurements of OHC bundle intensity in Tmc1^+/+ (closed bar) and Tmc1^Bth/Bth (hatched bar) for apical and basal OHCs. Numbers of cells measured are shown above the bars from three P6–P8 mice in each genotype. Error bars represent means ± SD.