Extrasynaptic localization of inactivating calcium-activated potassium channels in mouse inner hair cells

Abstract

Auditory hair cells from nonmammalian vertebrates are electrically tuned to specific sound frequencies primarily by the interactions of voltage-gated calcium channels and calcium-activated potassium (BK) channels colocalized at synaptic active zones. Mammalian inner hair cells are not electrically tuned and, yet, BK channels are also thought to reside at active zones. Using patch-clamp recordings and immunofluorescence, we characterized BK channel expression in mouse inner hair cells. Unexpectedly, these channels have inactivating currents and are clustered near the apex of the cell away from synaptic sites near the base. These results indicate a novel function of BK channels in mammalian inner hair cells and provide a framework for future research.

Figure 1.

IBTX isolates a fast activating and inactivating outward current from IHCs of mice with mature hearing. Outward currents in response to depolarizations from -60 to 40 mV in 10 mV steps were recorded from IHCs from intact mouse organs of Corti as described in Materials and Methods. A, B, Outward currents recorded from IHCs from P10 mice activate slowly (A) and are not affected by the addition of 100 nm IBTX in the extracellular solution (B). D, E, In contrast, outward currents recorded from IHCs from P20 mice show a rapidly activating component (D) that is blocked by the presence of 100 nm IBTX (E). C, F, Subtraction of the currents recorded in the presence of IBTX from the total currents recorded in control solution reveals little BK current in IHCs from P10 mice (C) and the presence of an inactivating BK current in IHCs from P20 mice (F). G, The time constants of activation for these currents are compared over a range of positive command potentials. H, The voltage dependence of inactivation of the BK current isolated from IHCs of P20 mice is shown over the same voltage range.

Figure 2.

Antibody staining against the BK channel reveals punctate staining on mIHCs. Intact organs of Corti from P20 mice were immunostained as described in Materials and Methods. A, Immunostaining with a monoclonal calretinin antibody (green) and polyclonal BK channel antibody (red) shows the organization of the hair cells into three rows of outer hair cells and one row of IHCs that additionally shows punctate staining for the BK channel. B, The monoclonal BK channel antibody recognizes the same protein as the polyclonal antibody as evidenced by colocalized immunoreactivity in samples immunostained with both the polyclonal (green) and monoclonal (red) BK channel antibodies. C, Preabsorption of either the polyclonal or monoclonal BK channel antibody abolishes immunoreactivity on the IHCs.

Figure 3.

Electrophysiology and antibody staining reveal a corresponding developmental increase in BK current and BK channel immunoreactivity. BK currents in response to depolarizations from -60 to 40 mV in 10 mV steps were isolated from recordings from mice IHCs of various ages as described in Materials and Methods. A, Little BK current is detected from IHCs from mice before the onset of hearing (P10), whereas the peak BK current increases in IHCs from mice after the onset of hearing (P14) and further increases in IHCs from mice with mature hearing (P20). C, The maximum BK current detected at each command potential is compared for the three developmental time points. B, Similarly, immunostaining with a polyclonal BK channel antibody shows little immunoreactivity in IHCs from P10 mice with increasing immunoreactivity just after the onset (P14) to the maturation (P20) of hearing. D, Comparison of the peak BK currents detected at 40 mV to the number of immunofluorescent puncta per field of view normalized to their respective values for P20 samples shows that the assessments of the developmental acquisition of the BK channel by both electrophysiology and immunoreactivity are comparable.

Figure 4.

Antibody staining reveals extrasynaptic localization of BK channels near the apex of mIHCs. Three-dimensional reconstructions of immunostained IHCs from P20 mice were generated from stacks of confocal micrographs as described in Materials and Methods. Various angles of rotation are shown for IHCs double immunostained with either a monoclonal calretinin antibody (green), to label IHCs, and a polyclonal BK channel antibody (red; A) or IHCs triple-immunostained with a polyclonal calretinin antibody (green), monoclonal NF200 antibody (isotype IgG₁; cyan), to stain afferent nerve fibers that contact the IHCs, and a monoclonal BK channel antibody (isotype IgG_2A; red; B). Reconstructions reveal extrasynaptic localization of BK channels near the apex of mIHCs.