Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Abstract

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca(2+) entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca(2+)-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca(2+) reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca(2+) concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca(2+) buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca(2+), intracellular Ca(2+) buffering, and membrane potential, by their common effect on intracellular free Ca(2+).

Fig. 1.

MET currents in mouse cochlear OHCs and IHCs. (A and B) Saturating MET currents recorded from an OHC (A) and an IHC (B) in response to 50-Hz sinusoidal force stimuli to the hair bundles at membrane potentials of −121 and +99 mV. The driver voltage (DV) signal of ±35 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The arrows and arrowheads indicate the closure of the transducer channels, i.e., disappearance of the resting current, during inhibitory bundle displacements at –121 mV and +99 mV, respectively. Dashed lines indicate the holding current. (C) Peak-to-peak MET current–voltage curves obtained from 14 OHCs and 7 IHCs (P6–P9) using 1.3 mM extracellular Ca²⁺. The fits through the current–voltage curves are according to a single-energy-barrier model (Eq. 1):$I\left(V\right)=k\left[\exp \left(\left(1-\gamma \right)\left(V-V_{\text{r}}\right)/V_{\text{s}}\right)-\exp \left(-\gamma \left(V-V_{\text{r}}\right)/V_{\text{s}}\right)\right],$where k is a proportionality constant, V_r is the reversal potential, V_s is a measure for the steepness of the rectification, and γ is the fractional distance within the membrane’s electrical field of an energy barrier, as measured from the outside (20). The values of the fits are as follows: OHCs: k = 444 pA, V_r = 1.3 mV, V_s = 38 mV, γ = 0.41; IHCs: k = 323 pA, V_r = 0.2 mV, V_s = 40 mV, γ = 0.42. (D) MET currents recorded at −81 mV (Lower) from an OHC elicited by force-step stimuli (Upper). Positive DVs of 50-ms duration (excitatory direction) elicited inward MET currents that declined or adapted over time (arrow in Right). Holding current: −90 pA. A small transducer current was present at rest, and inhibitory bundle displacements turned this off (gray traces). Upon termination of the inhibitory stimulus, the MET current showed evidence of rebound adaptation (arrowheads). Right shows double-exponential fit to the MET current decay (Results). The bundle movement for the inhibitory and excitatory step was −221 and 81 nm, respectively. In this and the following figures, bundle motion during fluid jet stimulation was estimated by using a conversion value of 10.1 nm/V obtained with a photodiode system (Materials and Methods and Fig. S2). (E) MET currents recorded at +99 mV from the same OHC as in D. Holding current: +861 pA. Note that all manifestations of transducer current adaptation [current decline during excitatory stimuli and rebound following inhibitory stimuli (D)] were absent at +99 mV, and the resting current was increased. (F) Normalized peak MET current recorded from seven OHCs at the holding potential of −81 mV and during a step to +99 mV as a function of bundle displacement. Note the leftward shift and shallower slope at +99 mV. The resting open probability between −81 mV (0.033 ± 0.004; n = 7) and +99 mV (0.22 ± 0.02) was found significantly different (paired t test; P < 0.0002). The relation between the normalized MET current and hair-bundle displacement was fitted by using a second-order Boltzmann function (Eq. 2):$I/I_{\max }=1/\left(1+\exp \left(a_2\left(x_2-x\right)\right)\ast \left(1+\exp \left(a_1\left(x_1-x\right)\right)\right)\right).$Fits to the data points were as follows: at –81 mV: I_max = −1,024 pA, a₁ = 0.044 nm⁻¹, a₂ = 0.015 nm⁻¹, x₁ and x₂ = 51 nm; at +99 mV: I_max = 1,744 pA, and the other parameters were as at −81 mV, except for x₁ = −10 nm, indicating a leftward shift of 61 nm. (G) MET currents recorded at −81 mV (Left) and +99 mV (Right) from an IHC elicited by force-step stimuli (Upper) as in D. Holding current: −132 pA (Left) and +1,268 pA (Right). Note that adaptation was absent at +99 mV.

Fig. 2.

Low extracellular Ca²⁺ removes MET current adaptation in mouse cochlear hair cells. (A) MET currents recorded from an OHC (Left) and an IHC (Right) in response to a 50-Hz sinusoidal force stimulus to the hair bundles at a membrane potential of −81 mV and in the presence of 1.3 mM extracellular Ca²⁺. DV, driver voltage signal. The arrows indicate closure of the transducer channels and dashed lines the holding current. (B) MET currents recorded at −81 mV from an OHC and an IHC in the presence of endolymphatic Ca²⁺ (0.04 mM) instead of 1.3 mM Ca²⁺. Note the larger resting MET current (difference between arrow and dashed line). (C) Resting open probability of the MET current obtained in OHCs and IHCs at −81 and +99 mV in 1.3 mM extracellular Ca²⁺ (Left) and at −81 mV in 0.04 mM extracellular Ca²⁺ (Right). The resting open probability was calculated by dividing the resting MET current (the difference between the current level before the stimulus, indicated by the dashed line, and the current level at the negative phase of the stimulus when all channels were closed) by the maximum peak-to-peak MET current. Number of cells is stated by the data points. (D) Step driver-voltages to the fluid jet (Upper) and MET currents recorded from an OHC at −81 mV in the presence of 0.04 mM extracellular Ca²⁺. Holding current: −1,378 pA. Note that all manifestations of MET current adaptation (Fig. 1D) are absent. (E) Normalized peak MET current recorded from six OHCs at the holding potential of −81 mV and during a step to +99 mV as a function of bundle displacement in the presence of low extracellular Ca²⁺ (0.04 mM). Note the absence of the leftward shift in the MET current-bundle displacement upon stepping V_m to +99 mV. The resting open probability between −81 mV (0.37 ± 0.04, n = 6) and +99 mV (0.40 ± 0.05) was not significantly different (paired t test). (F) Step driver-voltages and MET currents recorded from an IHC at −161 mV in the presence of 0.04 mM extracellular Ca²⁺. Holding current: −424 pA.

Fig. 3.

MET current adaptation in mouse cochlear hair cells is removed by high intracellular BAPTA. (A and B) MET currents recorded from an OHC (A) and an IHC (B) in response to a 50-Hz sinusoidal force stimuli to the hair bundles at the membrane potential of −81 mV in the presence of different intracellular BAPTA concentrations. Recordings are as in Fig. 1 A and B. Note the increased resting MET current (difference between dashed lines and arrows) with increasing BAPTA concentration. (C) Resting open probability of the MET current recorded in OHCs and IHCs obtained at −81 mV and using different concentrations of intracellular BAPTA. (D) Step driver voltages to the fluid jet (Top) and MET currents recorded from OHCs (Left) and IHCs (Right) at −81 mV (Bottom) and +99 mV (Middle) in the presence of 0.1 mM BAPTA. Note that all manifestations of MET current adaptation were removed at +99 mV as shown in Fig. 1 D and E with 1 mM EGTA. (E) MET currents recorded from OHCs (Middle) and IHCs (Bottom) at −81 mV in the presence of 5 mM BAPTA. (F) MET currents recorded from an OHC at −81 and +99 mV in the presence of 10 mM BAPTA. As for 5 mM BAPTA (E), MET current adaptation was absent. (G) Normalized peak MET current recorded from OHCs at the holding potential of −81 and +99 mV as a function of bundle displacement in the presence of 5 mM (n = 6; Left) and 10 mM (n = 6; Right) intracellular BAPTA. Similar to the experiments in low Ca²⁺ (Fig. 2E), little (5 mM) or no (10 mM) leftward shift in the MET current-displacement relation was observed when cells were held at +99 mV in the presence of intracellular BAPTA. Resting open probability between −81 mV (5 mM: 0.39 ± 0.02, n = 6; 10 mM: 0.43 ± 0.04, n = 6) and +99 mV (5 mM: 0.57 ± 0.02; 10 mM: 0.48 ± 0.04) was only significantly different using 5 mM BAPTA (paired t test: P < 0.001).

Fig. 4.

Strong Ca²⁺ buffering and depolarization abolish the adaptive shift in response to a paired-pulse protocol. MET currents recorded from OHCs elicited by paired-pulse stimulation (Upper). Steps 1 and 2 were 1.5 ms in duration and elicited maximal MET currents. Step 3, the adaptive step (81 nm), was 20 ms in duration and elicited nonsaturating current to move the bundle to the most sensitive region of the current-displacement curve (Fig. 1 D–G). MET current recorded in P6 OHCs at −81 mV (A) and +99 mV (B) in the presence of intracellular 0.1 mM BAPTA and at −81 mV in 10 mM BAPTA (C). Lower shows the normalized peak MET currents recorded in P6–P7 OHCs before (step 1) and during (step 2) the adaptive step (step 3) as a function of bundle displacement in 0.1 mM (A and B, n = 3) and 10 mM intracellular BAPTA (C; n = 7). The bundle displacement at which 50% of the maximum MET current is activated was found to be significantly different between step 1 (100.4 ± 15.0 nm; n = 3) and step 2 (132.2 ± 18.7 nm) in 0.1 mM BAPTA and at −81 mV (A: paired t test: P < 0.05). No significant difference was found in all of the other experimental conditions (B: step 1: 48.3 ± 8.9 nm, n = 3; step 2: 32.2 ± 6.3 nm; C: step 1: 71.6 ± 11.9 nm, n = 7; step 2: 60.7 ± 17.9 nm). Data points in A were fitted using Eq. 2: step 1 I_max = –841 pA, a₁ = 0.020 nm⁻¹, a₂ = 0.013 nm⁻¹, x₁ and x₂ = 67 nm; step 2 I_max = −812 pA, and the other parameters were as for step 1, except for x₁ = 141 nm, which show a Ca²⁺-dependent adaptive shift of 74 nm to the right, in agreement with previous finding in turtle hair cells (10).