Department of Physiology, University of Kentucky, Lexington, KY 40536-0298, USA.
J Assoc Res Otolaryngol. 2011 Dec;12(6):729-40. doi: 10.1007/s10162-011-0288-x. Epub 2011 Aug 31.
Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.
氨基糖苷类耳毒性涉及抗生素分子在内耳毛细胞中的积累，以及这些细胞的随后退化。氨基糖苷类物质在体内进入毛细胞的确切途径仍不清楚。与其他小有机阳离子类似，氨基糖苷类物质可以通过内吞作用带入细胞，或者通过大的非选择性阳离子通道（例如机械转导通道或 ATP 门控 P2X 通道）渗透。在这里，我们表明氨基糖苷类抗生素庆大霉素可以通过 TRPA1 进入小鼠外毛细胞（OHCs），TRPA1 是由某些刺激性化合物和脂质过氧化的内源性产物激活的非选择性阳离子通道。使用常规和穿孔全细胞膜片钳记录，我们发现 TRPA1 激动剂的应用会在野生型 OHCs 中引发内向电流反应，但在同源 Trpa1 敲除小鼠的 OHCs 中则不会。在转染了小鼠 Trpa1 的异源细胞中观察到了类似的与非选择性阳离子通道激活一致的反应。在用 TRPA1 激动剂短暂激活后，转染 Trpa1 的细胞会负载荧光氨基糖苷类抗生素 TexRed 缀合物（GTTR）。在未转染或未转染的细胞中未观察到这种摄取。在小鼠 Corti 器官外植体中，TRPA1 的激活导致 GTTR 和另一种小阳离子染料 FM1-43 快速进入 OHCs 和一些支持细胞，即使在钙离子游离溶液预孵育破坏毛细胞机械转导的情况下也是如此。在野生型小鼠中观察到这种由 TRPA1 介导的 GTTR 和 FM1-43 进入 OHCs，但在 Trpa1 敲除小鼠中则没有观察到，并且 PPADS（一种非选择性 P2X 通道阻断剂）不能阻断这种进入。值得注意的是，4-羟基壬烯醛（一种已知在氧化应激期间产生并在噪声暴露后在耳蜗中积累的内源性分子）激活了小鼠 OHCs 中的 TRPA1 通道。我们得出结论，TRPA1 通道可能为氨基糖苷类物质进入 OHCs 提供了一种新途径。
