Figure 3.
Domain 3, comprising the basolateral calyx and the stretch of axon leading to the heminode, selectively expressed NaV channels, KCNQ3, and certain scaffolding proteins. (A) Pan-NaV antibody produced a complex pattern of label, including Domains 1 and 3 (arrows) and the heminode (Domain 4), as well as type I hair cells (arrowheads) and adjacent type II hair cells (asterisk). (B, C) Pan-dystrophin (Pan-dystr, red) labeled Domain 3 of dimorphic (calretinin-negative), but not calyx-only (calretinin-positive, green) afferents (compare B with C1 and C2). (D) Domain 3 in peripheral-zone dimorphic afferents was intensely immunoreactive for KCNQ3, as shown by this flattened projection of a stack of confocal images. KCNQ3 staining (arrows) extended throughout Domain 3 from the basolateral outer surface of the calyx to the heminode. Note the lack of stain in Domain 2, marked by dashed white lines. The inset shows a higher magnification single image of a dimorphic afferent with a “complex” calyx ending that envelops two adjacent type I hair cells. Antibody to ezrin (green) labeled the apical microvilli of the sensory epithelium and the heminode, where it combined with the red KCNQ3 immunolabel to form a yellow band. (E) AnkyrinB antibody labeled Domain 3 in dimorphic afferents (long arrows) but not calyx-only afferents (arrowheads). Scale bars: A, D, E, 10 µm; D, inset, 5 µm; B, C, 2 µm.