Research ArticleHEALTH AND MEDICINE

Cooperativity of Kv7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells

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Science Advances  08 Apr 2020:
Vol. 6, no. 15, eaba1104
DOI: 10.1126/sciadv.aba1104
  • Fig. 1 Kv7.4 clusters increased in OHCs along the frequency-place map of the mammalian cochlear.

    (A) Representative segment of the mouse cochlea, showing the approximate frequency-place map from apical to basal contour (arrows). The black arrows indicate the predicted labeled sound frequencies (in kilohertz), represented on the cochlear map. Scale bar, 100 μm. (B to D) The expression pattern of Kv7.4 channels in OHCs from P4 (B), P8 (C), and P24 (D), respectively. Scale bar in (D) [representing (B) to (D)], 10 μm. The left panel represents two-dimensional (2D) confocal sections. Hair cells were labeled with myosin 7A antibody (Alexa Fluor 488 or fluorescein isothiocyanate in green) and Kv7.4 antibody (Alexa Fluor 555 in red). The middle and right panels show a 3D rendition of Z-stack images representing the front and back of the images, respectively, to locate Kv7.4 expression. Shown in the right panel are schematic representations summarizing channel expression during development. (E) By P24, when there are defined Kv7.4 clusters at the basolateral region of OHCs across the frequency-place map of the cochlea, semiquantitative analysis of the surface area of clusters was performed using the Imaris software routine and plotted against the apparent location and frequency representation of OHCs. The inset (above) shows examples of Kv7.4 clusters used to measure the areas shown in the bar graph. Scale bar, 4 μm. Data were generated from 50 OHCs from each frequency location and averaged from 12 different cochleae. Data were compared to OHCs at the apical tip of the cochlea (2 kHz) (***P < 0.001). (F) Kv7.4 fractional ratio was estimated as the area of Kv7.4 cluster/OHC surface area. The relation between the Kv7.4 cluster ratio along the frequency-place map of the cochlea was best described with a natural logarithmic function of the normalized frequency, from 2 to 10 kHz, where 10 kHz was set as 1. The data represent a onefold increase in channel fractional cluster ratio per octave.

  • Fig. 2 Outward K+ currents in mouse OHCs show sensitivity toward lino and a profound negative shift in the steady-state voltage-dependent activation and fast onset of activation time constant (τact) during development.

    The linopirdine (lino)–sensitive current (IKn) in OHCs increases in density and shows a smaller τact as Kv7.4 cluster density increases. (A and B) Currents elicited in response to voltage steps in P8 to P24 OHCs from mice were recorded from the cochlear location at CF of 3 to 4 kHz. Currents were elicited by applying voltage steps from −130 mV with increasing voltage steps in 10-mV increments. Deactivation voltage steps were at −30 mV. The sensitivity of the current was tested with 200 μM lino, and the difference current (in dotted plots) was fitted with one exponential function shown in solid lines to obtain time constants of activation (τact). (C) Current density (I)–voltage (V) relations fitted with a single Boltzmann function of the form I(V) = Imax/[1 + exp.(−(VV1/2))/k], where Imax is the maximum current density, V is the membrane voltage, V1/2 is the half-activation voltage, and 1/k is the voltage sensitivity at half-activation. The V1/2 (in mV) and 1/k (in V−1) for the currents at P8 and P24 were −33.6 ± 1.0 and 56.5 ± 5.1 (n = 15) and −81.1 ± 1.7 and 97.1 ± 6.0 (n = 17), respectively. Comparing V1/2 and 1/k at P8 and P24 currents (V1/2, P < 0.0001 and 1/k, P < 0.0001). (D) Relations between V1/2 of IKn in P24 OHCs at different cochlear locations, showing no visible changes (). The voltage sensitivity at half-activation of IKn increased relative to frequency by 18 V−1 kHz−1 () and Kv7.4 fractional area ratio () as a function of CFs of OHCs. (E) IKn voltage dependence in P24 mouse OHCs from different frequency-place maps of the cochlea, representing CFs (in kHz) 2, 4, 6, and 10. Relations between the maximum current density () measured in pA/pF, τact of currents at 0 mV (), and fractional area ratio () as a function of CFs are shown for comparison.

  • Fig. 3 Activation and deactivation kinetics of IKn in OHCs at different temperatures, cochlear locations, and CFs.

    (A) Family of IKn traces recorded from an OHC located at a 2-kHz frequency-place map of a 24-day-old mouse cochlea at room temperature (20°C, left), 30°C (middle), and physiological temperature (37°C, right). Currents were elicited from a holding voltage of −120 mV, and the voltage steps ranged from −120 to 50 mV with ΔV = 10 mV. The deactivation test voltage was at −30 mV (not shown). (B) Relations between activation time constants (τact) and voltages at 20°C (●), 30°C (●), and 37°C (●). The activation kinetics were fitted with a single exponential function [see insets in the right panel for examples of fitted traces of activation (top) and deactivation (bottom)]. Summary data were obtained from 3- to 4-kHz OHCs from five cochleae (n = 14). The data represent recordings in which three temperatures (20°, 30°, and 37°C) were successfully achieved from the same OHCs. The Q10 of IKn, which was expressed as (τ2τ1)(10T2T1) where T is temperature and τ is the time constant of activation, was 2.3. (C) Summary data for τact of IKn generated at a step voltage of −40 mV from OHCs at different CFs of the cochlea at 30°C (●); n = 27 cochleae. The number of OHCs for each data point is indicated (●), and the extrapolated τact at 37°C was obtained using Q10 = 2.3 (solid blue line).

  • Fig. 4 Induction of Kv7.4 clusters by CRY2-CIBN optogenetic actuator strategy mediates changes in voltage dependence and time dependence of activation of Kv7.4 currents.

    (A) Graphic representation of blue light (488 nm)–induced oligomerization system with CIBN fused to the N terminus of Kv7.4. (B) Time course of CIBN-EGFP-Kv7.4 channel cell surface and CRY2-mCherry expression probed using live-cell imaging. FRAP experiments were performed in HEK 293 cells transfected with CIBN-EGFP-Kv7.4 channel and CRY2-mCherry plasmids. Sequential images of cells and region of interest (ROI) are shown for control (C), bleach (B), and recovery (R) after bleach (insets represent enlarged ROI). Relative fluorescence intensity of the ROI and the time course of recovery for CIBN-EGFP-Kv7.4 channel (green) and CRY2-mCherry (red). Scale bar, 1 μm. Time constants of recovery (τ) were as follows: CIBN-EGFP-Kv7.4 = 1.4 ± 0.6 min (n = 9) and CRY2-mCherry = 1.0 ± 0.4 min (n = 9). HEK 293 cells transfected with EGFP (black) and mCherry (not shown) alone had faster τ of recovery = 0.2 ± 0.1 and 0.1 ± 0.2 min (n = 5). Comparing the recovery τ of CIBN-EGFP-Kv7.4 and EGFP, P < 0.001; and CRY2-mCherry and mCherry, P < 0.001. (C) Typical current records from HEK 293 cells expressing CIBN-GFP-Kv7.4 and CRY2-mCherry before (black traces) and after 4-min pulse of blue-light exposure (blue trace). Noticeably, induction of channel clusters sped up the time course of activation, but the current magnitude remained unchanged [maximum current density (pA/pF) before (72.8 ± 19.9) and after (74.5 ± 16.8) blue-light illumination, n = 18, P = 0.8]. (D) The voltage dependence of normalized current density (I/Imax). Black (●) and blue (●) solid circles represent data generated from current records before and after blue-light exposure. Fits (in solid lines) of the voltage dependence of activation, using single Boltzmann function yielded the following: before blue light (in mV), V1/2 = −15.1 ± 1.2, 1/k (V−1) = 66.2 ± 3.9 (n = 14); 5 min after blue light, −23.0 ± 1.1, 1/k = 95.2 ± 4.7 (n = 14). Comparing V1/2 and 1/k before and after blue-light exposure (V1/2, P < 0.0001 and 1/k, P < 0.0001). (E) Relations between activation time constant (τact), as a function of step voltages, show at least two- to eightfold reduction, depending on the voltage step, after blue-light exposure. (F) Summary data of blue-light exposure–time dependence of τact of CIBN-EGFP-Kv7.4 (○) and CIBN-EGFP-Kv7.4 + CRY2-mCherry (●) channels (n = 6 cells). The inset traces are representative of the summary data. Traces were elicited from −80-mV holding voltage and test voltage was 0 mV: [(in pA/pF) 48.4 ± 12.6 and 51.1 ± 17.2; n = 21, P = 0.6]. (G) Gating current records elicited in response to voltage steps from cells transfected with CIBN-EGFP-Kv7.4 and CRY2-mCherry. Gating currents were generated from a holding potential of −100 mV, using voltage steps ranging from −100 to 50 mV, ΔV = 10 mV. Integral of gating currents (Q) and the voltage dependence fitted by a single Boltzmann function before and after exposure to blue light. V1/2 (mV) before and after blue light was −40.9 ± 2.1 and −45.7 ± 2.4 (n = 7, P < 0.01), and the voltage sensitivity at half-activation 1/k (V−1) was 58.8 ± 4.1 and 93.6 ± 3.3 (n = 7, P < 0.001). (H) Nonstationary fluctuation analyses of gating current. Data from 200 consecutive gating current traces collected at 1-s intervals. The mean gating current as a function of variance plotted and fitted with the function σ2 = i.I − (I2/N), where σ2, i, I, and N represent variance, single gating charge current amplitude, macroscopic gating current, and the total number of channels, respectively. Estimates from the best fits for i and N before and after blue-light exposure were i = 0.06 ± 0.01 pA and 0.05 ± 0.02 pA (n = 4, P = 0.41), and N = 1177 ± 198 and 1002 ± 203 (n = 4, P = 0.26). The leftward shift in the fluctuation analyses suggests cooperativity postchannel clusters.

  • Fig. 5 CIBN-EGFP-Kv7.4 oligomerization and Kv7.4 consolidation in OHCs and mechanical sensitivity.

    (A) HEK 293 cells transfected with CIBN-EGFP-Kv7.4 for 48 hours. Outward CIBN-EGFP-Kv7.4 current traces (black) recorded from −60-mV holding potential, stepped from −100 to 50 mV, ΔV = 30 mV. Current traces after exposure to blue light for ~5 min (blue traces) and blue light + mechanical (mec) (0.6 μm) cell-body displacement (green traces). (B) Current density (in pA/pF), voltage relations of summary data from seven cells, are shown. (C) The tail current at −40 mV and the V1/2 (mV) and 1/k (V−1) for activation curves: control, 0.6 ± 3.2 and 21.3 ± 3.5; after blue light, −2.6 ± 3.4 and 51.7 ± 6.1; and blue light + mec displacement, 5.5 ± 3.5 and 64.1 ± 4.4 (n = 7). Comparing V1/2, there was significant difference at the P < 0.05 level for the population means F(2,18) = 10, P = 0.001. Post hoc comparisons test for control and blue light (P = 0.2). Blue light + mec displacement versus control means were significantly different (P = 0.04). Blue light + mec displacement versus blue-light means were significantly different (P = 0.001). Comparing 1/k at the P < 0.05 level for the population means F(2,18) = 148, P = 0.0001. Post hoc comparisons using the Tukey HSD test indicated that all three condition means were significantly different (P = 0.0001). (D to F) Results similar to that described in (A) to (C); HEK 293 cells were cotransfected with CIBN-EGFP-Kv7.4 and CRY2-mCherry. (D) τact abbreviated by ~2-fold after 5-min blue-light exposure (at 60 mV), and current density increased after 0.6-μm displacement (~1.3-fold). The tail current at −40 mV, and the V1/2 (mV) and 1/k (V−1) for activation curves were as follows: control, 0.6 ± 1.4 and 55.5 ± 10.8; after blue light, −4.5 ± 3.2 and 52.0 ± 9.7; and blue light + mec displacement, −32.6 ± 2.7 and 60.3 ± 8.1 (n = 5). Comparing V1/2 at the P < 0.05 level for the population means F(2,12) = 250, P = 0.0001. Post hoc comparisons using the Tukey HSD for three conditions (P = 0.2). (G) Family of IKn traces (black) recorded from an OHC located at the 2-kHz frequency-place map of P6 mouse cochlea and after displacement of the basolateral cell wall by ~0.4 μm (green). Currents elicited from a holding voltage of −60 mV and the voltage steps from −130 to 50 mV, ΔV = 40 mV. (H) Similar recording as in (G) from P21 OHC. A displacement of 0.4 μm sped up the τact [at a 60-mV step, τact (ms) was 7.6 ± 4.9 and 0.9 ± 0.5 (n = 5, P < 0.05)] before and after mec displacement, respectively. (I) At P21, mec displacement produced a leftward shift in the steady-state activation properties. The tail current at −40 mV and the V1/2 (mV) and 1/k (V−1) for activation curves were as follows: control, −81.8 ± 0.9 and 36.6 ± 7.7, and mec displacement, −97.3 ± 2.8 and 60.6 ± 12.4 (n = 5). Comparing V1/2 (P < 0.0001) and 1/k (P = 0.006). Thus, 0.4-μm mec displacement produced a ~15 leftward shift in the voltage dependence and ~8-fold change in the τact in IKn. (J) IKn traces (black) from −60-mV holding voltage and voltage steps from −110 to 50 mV from P24 OHCs. In red traces are recordings from the same OHC ~5 min after bath perfusion of 630 mosmol (mannitol-based) solution. The current magnitude plummeted in five of seven OHCs. For the remaining two OHCs, the current magnitude was unchanged after hypertonic solution. (K) The summary data from five OHCs are shown as inset in the current-voltage relationship. V1/2 for IKn in P24 OHC was −73.2 ± 3.3 mV; the voltage sensitivity at half-activation, 1/k, was 79.4 ± 9.3 V−1 (n = 5); posthypertonic solution (2×) was −59.3 ± 3.7 mV and 1/k was 68.5 ± 8.8 V−1 (n = 5). Comparing V1/2 (P = 0.0002) and 1/k (P = 0.09). (L) Changes in the τact relative to step voltages for IKn in normal and hypertonic solution (n = 5 OHCs).

  • Fig. 6 Biophysical model and potential contributions of Kv7.4 to OHC function.

    (A) Four-state chemical kinetic model of Kv7.4 gating of conductance based on Eyring's transition state theory (see the Supplementary Materials). The model accounts for clustering through entropy of the first transition, voltage gating through two transitions, and mechanical gating through enthalpy of the final transition. (B) The Kv7.4 conductance is inserted into a passive HEK model or an active piezoelectric model of the OHC (F). (C and D) Simulated voltage-clamp currents in Kv7.4-transfected HEK cells under control, oligomerized, and stress-relieved conditions. (E) Simulated voltage-clamp currents and activation kinetics based on OHC parameters (table S1). Effects of temperatures 20°C (black) and 37°C (red) on time constant of channel openings (τo). (F) The mechanical portion of the OHC model with an external load matched to the cell. (G) OHC tuning curves for a model cell from the 3-kHz region of the cochlea in the control condition (Null), after adding a DC Kv7.4 conductance (blue dashed line), and after adding mechanovoltage-sensitive Kv7.4 conductance (red solid line). (H) The power efficiency of the OHC is predicted to be increased by mechanovoltage-sensitive Kv7.4 (red solid line) while reduced by a passive DC conductance. (I) Schematic diagram showing conditions of an OHC at low (left) and high (right) frequency. At high frequency, viscous mass predominates, and there is a 180° phase switch resulting mainly from the mechanical gating of the Kv7.4 channel. IMET, mechanoelectrical transducer current. IKv7.4, Kv7.4 current.

Supplementary Materials

  • Supplementary Materials

    Cooperativity of Kv7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells

    Maria C. Perez-Flores, Jeong H. Lee, Seojin Park, Xiao-Dong Zhang, Choong-Ryoul Sihn, Hannah A. Ledford, Wenying Wang, Hyo Jeong Kim, Valeriy Timofeyev, Vladimir Yarov-Yarovoy, Nipavan Chiamvimonvat, Richard D. Rabbitt, Ebenezer N. Yamoah

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