Resting potential and submembrane calcium concentration of inner hair cells in the isolated mouse cochlea are set by KCNQ-type potassium channels

Abstract

Cochlear inner hair cells (IHCs) transduce sound-induced vibrations into a receptor potential (RP) that controls afferent synaptic activity and, consequently, frequency and timing of action potentials in the postsynaptic auditory neurons. The RP is thought to be shaped by the two voltage-dependent K+ conductances, I(K,f) and I(K,s), that are carried by large-conductance Ca2+- and voltage-dependent K+ (BK)- and K(V)-type K+ channels. Using whole-cell voltage-clamp recordings in the acutely isolated mouse cochlea, we show that IHCs display an additional K+ current that is active at the resting membrane potential (-72 mV) and deactivates on hyperpolarization. It is potently blocked by the KCNQ-channel blockers linopirdine and XE991 but is insensitive to tetraethylammonium and 4-aminopyridine, which inhibit I(K,f) and I(K,s), respectively. Single-cell PCR and immunocytochemistry showed expression of the KCNQ4 subunit in IHCs. In current-clamp experiments, block of the KCNQ current shifted the resting membrane potential by approximately 7 to -65 mV and led to a significant activation of BK channels. Using BK channels as an indicator for submembrane intracellular Ca2+ concentration ([Ca2+]i), it is shown that the shift in IHC resting potential observed after block of the KCNQ channels leads to an increase in [Ca2+]i to values > or =1 microm. In conclusion, KCNQ channels set the resting membrane potential of IHCs in the isolated organ of Corti and thus maintain [Ca2+]i at low levels. Destabilization of the resting potential and increase in [Ca2+]i, as may result from impaired KCNQ4 function in IHCs, provide a novel explanation for the progressive hearing loss (DFNA2) observed in patients with defective KCNQ4 genes.

Fig. 1.

Membrane potential of IHCs is set by a conductance sensitive to the KCNQ-channel blocker linopirdine. A, Current-clamp recording (at zero current) from an IHC. Application of the BK blocker TEA (5 mm) had no effect onV_R. In contrast, 10 μmlinopirdine depolarized V_R in a slowly reversible manner. Note the readily reversible depolarization by TEA in the presence of linopirdine. Application of increased extracellular K⁺ concentration at the end of the experiment is shown to illustrate the speed of solution exchange. B, Block of BK-mediated I_K,f currents of an IHC by extracellular TEA (5 mm). The IHC was voltage clamped at −84 mV and stepped to voltages between −74 and 16 mV in 10 mV increments [residual R_s, 0.15 MΩ (control) and 0.3 MΩ (TEA)].

Fig. 2.

Isolation of a voltage-dependent K⁺ current activated around the IHC resting potential. A, The fast and slow outward current components I_K,f andI_K,s (see Introduction) recorded in an IHC in response to the voltage protocol shown (voltage increment was 10 mV; residual R_s was 0.5 MΩ). Zero current level is indicated by a horizontal bar.B, Current response of the same IHC to the voltage protocol indicated; the experiment was done with 5 mm TEA present extracellularly to block I_K,f. Note that the recorded current was almost completely activated at the resting potential of approximately −70 mV and deactivated on hyperpolarization. Activation of I_K,soccurred only at voltages positive to −50 mV, as apparent in the top trace. Traces are shown with leak current (65 pA at −120 mV) subtracted. Zero-current level is indicated by thedotted line. C, Activation curve of the novel current recorded in B. Tail current amplitude was taken 1.5 msec after stepping to −124 mV, and prepulse duration at the various potentials was 500 msec to ensure steady-state conditions.Continuous line represents fit of a Boltzmann function (see Materials and Methods) to the normalized currents averaged from six IHCs; values for V_h and slope as yielded by the fit were −84.3 and 10.1 mV, respectively. D, Reversal potentials of the novel current measured at different extracellular K⁺ concentrations closely matched the K⁺ equilibrium potential given by the Nernst equation (straight line). Reversal potential was determined from leak-corrected instantaneous currents after steps to potentials between −144 and −54 mV. Leakage conductance (1.2 ± 0.3, 1.0 ± 0.2, and 1.3 ± 0.5 nS for 2, 5.8, and 12 mm K⁺_ex, respectively) was determined by a linear fit to currents remaining after complete deactivation of the KCNQ-type current at potentials between −144 and −124 mV. Extracellular TEA (5 mm) was used to block I_K,f. Data for 2, 5.8, and 12 mm K⁺_ex are mean ± SD from 4, 9, and 5 IHCs, respectively.

Fig. 3.

The IHC resting K⁺ current is sensitive to the KCNQ-channel blocker linopirdine. A, KCNQ current recorded in IHCs in response to voltage steps from −64 to −144 mV before and after application of linopirdine at the concentrations indicated. Extracellular [K⁺] was 20 mm throughout these experiments to increase the KCNQ current amplitude; dotted lines indicate zero-current level. B, Time course of linopirdine block shown for the cell in A. Symbols indicate amplitude of the transient inward current in response to each of the repetitive hyperpolarizing pulses. C, Linopirdine dose-inhibition curve of the KCNQ current measured as in A andB. Continuous line is a fit of the Hill equation to the data (see Results). Each data point represents mean ± SD of four to eight IHCs.

Fig. 4.

KCNQ4 immunoreactivity and single-cell PCR analysis of cochlear IHCs. A, Left, Confocal immunofluorescence image of the organ of Corti (midbasal turn, 6-week-old mouse) stained with the rabbit anti-KCNQ4 antibody (red) and DAPI (nuclear staining,blue). Right, Differential interference contrast image of the cryosection shown in A. Scale bar, 20 μm. B, C, Right, Cryosections as in A stained with the goat anti-KCNQ4 antibody (green) in the absence (B) or presence (C) of the antigenic peptide (see Materials and Methods) imaged by conventional immunofluorescence microscopy (midbasal turn, 4-week-old mouse); other fluorescence signals are from the anti-synaptophysin antibody (red) and DAPI. Scale bar, 20 μm.Left, Enlarged view of the IHCs shown atright. Scale bar, 10 μm. Note that KCNQ4 immunoreactivity is found in both OHCs and IHCs with an intense staining at the basal pole of OHCs (indicated by arrows) and a weaker and nonuniform signal in IHCs. D, Agarose gel analysis of the multiplex single-cell RT-PCR performed in the cell types indicated (DC, Deiters' cell); GAPDH was used as a control for successful isolation of mRNA. Co indicates the control PCR performed with extracellular fluid collected directly adjacent to IHCs.

Fig. 5.

Voltage dependence of BK currents in IHCs.A, BK currents (I_K,f) recorded in isolation using 10 mm 4-AP in the pipette and 1 μm linopirdine in the extracellular medium in response to the voltage protocol indicated (residualR_s, 0.35 MΩ). B, Voltage dependence of BK currents measured with the tail current protocol indicated. Voltage steps to the various potentials were kept as short as possible (3 msec) to avoid artifacts caused by K⁺ accumulation near the membrane resulting from the large outward currents (residual R_s, 0.3 MΩ). C, Tail currents from the experiment inC shown at enlarged scales. D,G–V relationship obtained from experiments as inB with residual R_s ≤0.35 MΩ. Tail current amplitudes were taken 0.2 msec after the step to −49 mV and normalized to current amplitude at saturation (I_max) obtained from a Boltzmann fit to the current–voltage relationship for each cell. Continuous line shows a Boltzmann fit to the averaged data (mean ± SD of 6 experiments), yielding values for V_hand α of −44.6 and 8.9 mV, respectively.

Fig. 6.

Ca²⁺ dependence of BK currents in inside-out patches reveals a voltage-dependent increase of [Ca²⁺]_i in the intact IHC.A, BK currents measured in response to depolarizing voltage steps in an inside-out patch excised from an IHC at the [Ca²⁺]_i indicated. Voltage protocols were as follows: left (0 and 1 μmCa²⁺), holding potential (V_H), −104 mV; voltage steps, −104 to +136 in 20 mV increments; tail current voltage (V_T), +26 mV; right (3 and 10 μm Ca²⁺),V_H, −104 mV; voltage steps, −144 to +116 mV in 20 mV increments; V_T, +11 mV. Each trace is averaged from 20 individual current recordings; 10 mm 4-AP was present in all solutions to block other K⁺ currents. B, G–Vrelationships of IHC BK channels at 0, 1, 3, and 10 μmCa²⁺ obtained from 21, 15, 11, and 12 patches using tail current analysis as described in Figure 5D.Symbols refer to the different [Ca²⁺]_i indicated in A.C, Overlay of BK activation curves as obtained from patch and whole-cell measurements. Curves shown are fits to the data replotted from Figure 5D [whole-cell (WC), black line] and fromB (inside-out patch, gray lines). Note that activation of BK currents in the intact cell displays a substantially steeper voltage depen dence than when recorded with constant [Ca²⁺]_i. D, BK activation near the resting potential of the IHC shown at enlarged scale. Curvelabeled with an asterisk is the fit to the individualG–V relationship at 1 μmCa²⁺ that yielded the most negativeV_h (−11 mV). Intersection of whole-cell activation curve with mean activation at 3 μm occurs at approximately −59 mV, and with the leftmost activation curve at 1 μm Ca²⁺ occurs at approximately −64 mV, indicating a whole-cell [Ca²⁺]_i of 1–3 μm at −65 mV.