BK channels mediate cholinergic inhibition of high frequency cochlear hair cells

Abstract

Background: Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K(+) channels that control the membrane potential and also ligand-gated K(+) channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.

Methodology/principal: Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K(+) currents.

Conclusions/significance: Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.

Figure 1. Tonotopic differences in outer hair cell properties.

(A) Micrograph showing a P21 rat cochlea in which the bony covering has been removed to expose the intact organ of Corti. The overlayed dashed arrows indicate the progression from low frequency apical turns to higher frequency basal turns. The locations of the apical and basal regions of the organ of Corti (and their approximate frequency ranges) that were examined in this investigation are indicated on black. DIC micrographs of isolated whole mount preparations of apical (B) and basal (D) regions of the organ of Corti show the sensory hair cells organized into a single row of inner hair cells (open arrowhead) and three rows of outer hair cells (solid arrowheads). DIC micrographs of horizontally oriented outer hair cells from apical (C) and basal (E) regions reveal the typical decrease in outer hair cell length from apical (∼50 µm) to basal regions (∼20 µm). Representative records of currents evoked by a series of voltage steps (100 ms in duration) from −131 mV to 49 mV in 10 mV increments (with an interpulse holding potential of −81 mV) show an increase in outward current (at 49 mV) from 2.08±0.09 nA in outer hair cells from apical regions (n = 25, F) to 7.24±0.4 nA in outer hair cells from basal regions (n = 23 basal, G). All scale bars equal 10 µm.

Figure 2. BK channels do not contribute to voltage-gated K⁺ currents in apical outer hair cells.

(A) Whole-cell currents recorded from an apical outer hair cell in response to voltage steps (100 ms in duration) from −131 mV to 49 mV in 10 mV increments (with an interpulse holding potential of −81 mV) before (black line) and in the presence (grey line) of bath application of 100 nM IBTX, a specific blocker of BK channels, show little IBTX-sensitive currents. (For clarity only some of the traces are shown.) (B) No differences in voltage-gated currents were observed in response to IBTX application across all test potentials examined in apical outer hair cells. (C) Bar plot comparing the fractional contribution of IBTX- and linopirdine-sensitive currents to apical outer hair cells at 49 mV show that apical outer hair cells express predominantly linopirdine-sensitive KCNQ4 currents and little or no BK currents. Confocal micrograph (1 optical section) of an apical cochlear turn immunostained with a monoclonal antibody against the BK channel (red, D and F) shows immunoreactivity in the region corresponding to the neck of the inner hair cells (indicated with an open arrowhead) but no immunoreactivity in the region associated with the outer hair cells (indicated by the solid arrowheads), immunostained with a polyclonal antibody against prestin (green, E and F). Scale bar equals 10 µm.

Figure 3. KCNQ4 and BK channels contribute to voltage-gated K⁺ currents in basal outer hair cells.

(A) Whole-cell currents recorded from a basal outer hair cell in response to voltage steps (100 ms in duration) from −131 mV to 49 mV in 10 mV increments (with an interpulse holding potential of −81 mV) before (black line) and in the presence of either 100 µM linopirdine (red trace) or both 100 µM linopirdine and 100 nM IBTX (blue trace). (For clarity only 2 potentials, −131 mV and 49 mV are shown.) (B) At depolarizing membrane potentials, both linopirdine-sensitive KCNQ4 and IBTX-sensitive currents contribute to the voltage gated currents in basal outer hair cells. (C) Bar plot comparing the percent block of I_Max at 49 mV in apical and basal OHCs in the presence of 100 µM linopirdine (black bars) or both 100 µM linopirdine and 100 nM IBTX (grey bars). (D) Confocal micrograph (1 optical section) of a basal cochlear turn immunostained with a monoclonal antibody against the BK channel (red, D and F) shows immunoreactivity in the region corresponding to the neck of the inner hair cells (indicated with an open arrowhead) as well as immunoreactivity in the outer hair cells (indicated by the solid arrowheads), immunostained with a polyclonal antibody against prestin (green, E and F). Scale bar equals 10 µm.

Figure 4. Tonotopic distribution of BK channels in outer hair cells.

3D renderings of confocal z-stacks of apical (A), middle (B), and basal (C) cochlear turns immunostained with a monoclonal antibody against the BK channel (red) and a polyclonal antibody against synapsin (green) to label the efferent presynaptic terminals show that BK channel immunoreactivity in the three rows of outer hair cells (when present) is associated with efferent terminals. (D) Moreover, the size of BK channel immunopuncta increases from apical to middle and basal turns, paralleling an increase in size and likely number of efferent terminals. Data are presented as box plots representing the median (box interior), the 25th and 75th percentile (box boundaries), the 10th and 90th percentile (whiskers), and the 5th and 95th percentile (dots). (E) 3D renderings of confocal z-stacks of a middle cochlear turn immunostained with a polyclonal antibody against the BK channel (red) and a monoclonal antibody against the NaK-ATPase α3 (green) to label the efferent presynaptic terminal membrane shows adjacent but not co localized expression of the BK channel with the presynaptic efferent terminal membrane. The location of the three rows of outer hair cells are indicated (solid arrowheads). Grid dimensions equal 10 µm.

Figure 5. Tonotopic distribution of SK2 channels in outer hair cells.

3D renderings of confocal z-stacks of apical (A), middle (B), and basal (C) cochlear turns immunostained with a polyclonal antibody against the SK2 channel (green) show expression of the SK2 channel in the three rows of outer hair cells in all regions. Confocal z-stacks (23 optical sections) of basal cochlear turns immunostained with a polyclonal antibody against the SK2 channel (green, D) and monoclonal antibody against the BK channel (red, E) show colocalized expression (F) in the three rows of outer hair cells, further suggesting the localization of BK channels to the postsynaptic outer hair cell membrane. (Examination of single optical sections also shows colocalization of the SK2 and BK channel.) The size of the SK2 channel immunopuncta increases from apical to middle turns and then decreases in basal turns. Data are presented as box plots representing the median (box interior), the 25^th and 75^th percentile (box boundaries), the 10^th and 90^th percentile (whiskers), and the 5^th and 95^th percentile (dots). (G) 3D renderings of confocal z-stacks of basal cochlear turns immunostained with a polyclonal antibody against the SK2 channel (green), a monoclonal antibody against the BK channel (red), and a goat polyclonal antibody against synapsin (blue) confirm the colocalized expression of SK2 and BK channels in association with efferent terminals (H) in the three rows of outer hair cells. In basal turns, BK and SK2 channel immunoreactivity are colocalized. Grid dimensions equal 10 µm for top and bottom panels. Scale bar equals 10 µm for middle panels.

Figure 6. Tonotopic contribution of SK2 and BK channels at the outer hair cell-efferent synapse.

(A) Whole-cell currents recorded from an apical outer hair cell (OHC) voltage clamped at −34 mV in response to a short (100 ms) application of 1 mM ACh (time point indicated by arrow) before (control) and after application of either 100 nM IBTX (black) or 300 nM apamin (grey). (B) Bar plot comparing the fractional contribution of IBTX- and CHTX-sensitive BK and apamin-sensitive SK2 currents evoked in response to either 100 ms or 1 s application of ACh show that ACh-evoked currents in apical outer hair cells are predominantly mediated by SK2 channels. (C) Whole-cell currents recorded from a basal outer hair cell (OHC) voltage clamped at −51 mV in response to a short (100 ms) application of 1 mM ACh (timepoint indicated by arrow) before (control) and after application of either 100 nM IBTX (black) or 300 nM apamin (grey). (D) Bar plot comparing the fractional contribution of apamin-sensitive SK2 currents in apical and basal outer hair cells and IBTX-sensitive BK currents in basal outer hair cells in response to application of ACh show that ACh-evoked currents in basal outer hair cells are predominantly mediated by BK channels and not SK2 channels.