Ca(2+) and K(+) (BK) channels in chick hair cells are clustered and colocalized with apical-basal and tonotopic gradients

Abstract

Electrical resonance is a mechanism used by birds and many vertebrates to discriminate between frequencies of sound, and occurs when the intrinsic oscillation in the membrane potential of a specific hair cell corresponds to a specific stimulus sound frequency. This intrinsic oscillation results from an interplay between an inward Ca(2+) current and the resultant activation of a hyperpolarizing Ca(2+)-activated K(+) current. These channels are predicted to lie in close proximity owing to the fast oscillation in membrane potential. The interplay of these channels is widespread in the nervous system, where they perform numerous roles including the control of synaptic release, burst frequency and circadian rhythm generation. Here, we used confocal microscopy to show that these two ion channels are clustered and colocalized in the chick hair cell membrane. The majority of Ca(2+) channels were colocalized while the proportion of colocalized BK channels was markedly less. In addition, we report both an apical-basal gradient of these clusters in individual hair cells, as well as a gradient in the number of clusters between hair cells along the tonotopic axis. These results give physical confirmation of previous predictions. Since the proportion of colocalized channels was a constant function of Ca(2+) channels, and not of BK channels, these results suggest that their colocalization is determined by the former. The molecular mechanisms underpinning their clustering and colocalization are likely to be common to other neuronal cells.

Figure 1. Clustering and colocalization of Ca²⁺ and BK channels in hair cells

The figure shows a confocal image of hair cells from the chick approximately 30% fractional distance from the low frequency end of the papilla. A shows cells that have been labelled with an antibody to the BK channels (Slo) that cluster on the peripheral surface of the cell. B is the identical section showing labelling of clustered Ca²⁺ channels, also on the peripheral surface of the cell, identified by their size and intense fluorescence, with some clusters colocalizing with BK channel clusters. C is the merged image showing colocalized channel clusters (white arrows). A number of Slo channel clusters do not colocalize with Ca²⁺ channels (blue arrows). Line scan maps of the white line passing through the upper part of A and B are shown beneath each panel. The threshold used to demarcate cluster intensity from background is shown in the line scan map (black line). The scale bar is 10 μm.

Figure 2. There is an apical–basal gradient of ion channel clusters within a hair cell

The figure shows confocal images taken at varying depths in a number of hair cells (60% fractional distance from the low frequency end of the papilla) that have been labelled with antibodies to the BK (Rhodamine red) and Ca²⁺ (Fluorescein green) channels. Colocalized channels of these merged images are yellow. A is from the upper apical portion of these cells, B is from the middle and C is from the basal end. As is evident, the number of BK channels (red), Ca²⁺ channels (green) and colocalized channels (yellow) increase from the apical to basal end. The scale bar is 10 μm. D shows a bar graph of the mean number of BK channel clusters, Ca²⁺ channel clusters, and colocalized channel clusters in 5 segments of hair cells divided in an apical-to-basal direction. These 14 cells were obtained from three cochleas, and were located approximately 75% fractional distance from the low frequency end of the papilla. As is evident, the number of channel clusters is most concentrated at the base of the cell (P < 0.001 for both channels and colocalized channel clusters compared with any of the upper segments). Approximately half of a cell's ion channel clusters are located in the lowest segment.

Figure 3. There is a tonotopic gradient in ion channel clusters

Confocal images of hair cells from the low frequency (A) and high frequency (B) end of the basilar papilla that have been labelled with antibodies to the BK (red) and Ca²⁺ (green) channels is shown. The figure shows that the number of both channel clusters and colocalized channel clusters (yellow) is increased in cells in the high frequency location in the basilar papilla. The sections shown were taken across the basal fifth of the cells. The scale bar is 10 μm. C shows the mean number of ion channel clusters per cell (Colocalized/Ca²⁺/BK) from low frequency (15% fractional distance, 28 cells), mid-frequency (65% fractional distance, 28 cells) and high frequency (80% fractional distance, 23 cells) hair cells in bar graph form. The cells were from three cochleas. The numbers of each ion channel and colocalized channel clusters were significantly different in the cells from each of the three different frequency locations (P < 0.05). While the proportion of colocalized Ca²⁺ channels remained constant (∼80%), the proportion of colocalized Slo channels increased from 35% in low frequency hair cells to 58% in high frequency hair cells.