Figure 4

BK channel clusters are associated with the hair cell membrane. (A) Isolated hair cells were imaged using confocal microscopy to determine whether anti-BK clusters were associated with the membrane or found intracellularly. Two exemplar hair cells are shown, both oriented with hair bundles toward the top of the image. Between 13 and 17 optical sections were taken at increments of 0.5 μm. Projections of full Z-stack are shown alongside orthogonal cuts through the reconstructed stack. Cuts were made at the level of each anti-BK puncta and positioned to the right of each hair cell example. In all cases, puncta were present along the outer edge of the cut-view, indicating overlap with the hair cell plasma membrane. (B) Hair cells and auditory neurons were acutely isolated onto the same slide in some preparations and stained for glutamate receptor 2 (GluR2) in order to detect residual afferent terminals on the dissociated hair cells. Afferent auditory neurons were identified according to cell size and the presence of bipolar axonal projections. Under epifluorescence and using constant exposure conditions, anti-GluR2 label was found on the cell soma of auditory neurons but was absent from hair cells. (C) Anti-BK puncta were distant from synaptic ribbons in isolated hair cells. Isolated hair cells were co-labeled with anti-BK (red) and anti-ctbp2/RIBEYE (green), a marker for presynaptic ribbons. An exemplar tall hair cell is shown using epifluorescence imaging. Debris that might be associated with afferent terminals is absent from the matching differential interference contrast image (left). The image shown is representative of over 5 tall hair cells imaged in this co-labeling experiment. Scale bars in all panels = 10 μm.