Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, California, USA.
PLoS One. 2013;8(1):e54794. doi: 10.1371/journal.pone.0054794. Epub 2013 Jan 24.
Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.
声音感知需要功能性毛细胞机械转导（MET）机制，包括 MET 通道和尖端连接蛋白。先前的工作表明，耳毒性氨基糖苷类（AG）进入毛细胞需要功能性 MET 通道。在这项研究中，我们研究了尖端连接蛋白，包括钙粘蛋白 23（Cdh23），是否调节 AG 进入毛细胞。通过对耳蜗外植体进行延时显微镜检查，我们发现从 Cdh23(+/+)和 Cdh23(v2J/+)小鼠的三天大的毛细胞中快速摄取了结合了庆大霉素的 Texas Red（GTTR），但未能在 Cdh23(v2J/v2J)毛细胞中检测到 GTTR 摄取。用钙螯合剂 1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）预处理野生型耳蜗以破坏尖端连接，也有效地减少了 GTTR 进入毛细胞。Cdh23(v2J/v2J)和 BAPTA 处理的毛细胞均免受庆大霉素引起的变性。与对照相比，BAPTA 处理后 6 小时 GTTR 摄取仍然减少；24 小时后，与未处理的毛细胞相比，药物摄取无差异，而再次变得易受庆大霉素诱导的细胞死亡影响。这些结果共同提供了遗传和药理学证据，表明尖端连接对于毛细胞中的 AG 摄取和毒性是必需的。由于尖端连接可以自发再生，因此它们的暂时断裂为毛细胞免受 AG 毒性提供了有限的时间窗口。
