The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.
The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.
Furness
D N
DN
Department of Communication and Neuroscience, Keele University, Staffs, UK.
Hackney
C M
CM
Benos
D J
DJ
D000584:Amiloride / Q000633:toxicity*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000633:toxicity*; D001666:Binding Sites, Antibody / Q000187:drug effects; D004096:Dihydrostreptomycin Sulfate / Q000633:toxicity*; D004232:Diuretics / Q000633:toxicity*; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D006168:Guinea Pigs; D006199:Hair Cells, Auditory, Inner / Q000187:drug effects / Q000276:immunology / Q000648:ultrastructure; D018072:Hair Cells, Auditory, Outer / Q000187:drug effects / Q000276:immunology / Q000648:ultrastructure; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D007668:Kidney / Q000276:immunology / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D009925:Organ of Corti / Q000187:drug effects* / Q000276:immunology / Q000648:ultrastructure; D015398:Signal Transduction / Q000187:drug effects; D015222:Sodium Channels / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004232:Diuretics; D007106:Immune Sera; D015222:Sodium Channels; D000584:Amiloride; D004096:Dihydrostreptomycin Sulfate