Furness D N, Hackney C M, Benos D J
Department of Communication and Neuroscience, Keele University, Staffs, UK.
Hear Res. 1996 Apr;93(1-2):136-46. doi: 10.1016/0378-5955(95)00224-3.
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.
毛细胞上的机械电转导通道被认为是由纤毛顶部连接丝操控的，当毛束向最高一排静纤毛的方向偏转时，连接丝会被拉伸。因此，确定这些通道的位置是对这一假说的重要验证。已知转导通道对氨氯地平敏感，用针对肾脏中氨氯地平敏感的上皮钠通道（α NaCh）产生的抗体进行免疫金标记表明，具有相似特征的位点位于较短静纤毛的尖端似乎与相邻较高静纤毛的侧面接触的区域，而不是直接与纤毛顶部连接丝相关联。现在，已经进行了进一步的免疫细胞化学实验，以确定氨氯地平和二氢链霉素（二者均可阻断转导）是否会影响这种标记。当用α NaCh固定并孵育柯蒂氏器的表面制剂，然后加入适当的二抗时，可获得静纤毛的免疫荧光标记。这种标记在固定前用胰蛋白酶处理后会消失，但如果组织先用氨氯地平预处理，然后在其存在的情况下用胰蛋白酶处理，则标记会保留。由于已知氨氯地平可保护氨氯地平结合位点不被胰蛋白酶降解，这些结果表明α NaCh揭示了静纤毛上的氨氯地平结合位点。同样，如果耳蜗组织先用二氢链霉素（DHS）预处理，并在与α NaCh孵育之前在其存在的情况下固定，则静纤毛的免疫荧光标记会消失。使用透射电子显微镜对胶体金标记进行定量分析表明，DHS处理会使静纤毛上的金颗粒数量显著减少，尤其是在它们之间的接触区域。这些结果表明，抗Na⁺识别的位点具有与机械电转导通道相似的特征。
