Kirkwood Nerissa K, O'Reilly Molly, Derudas Marco, Kenyon Emma J, Huckvale Rosemary, van Netten Sietse M, Ward Simon E, Richardson Guy P, Kros Corné J
Sussex Neuroscience, School of Life Sciences, University of SussexBrighton, United Kingdom.
Sussex Drug Discovery Centre, School of Life Sciences, University of SussexBrighton, United Kingdom.
Front Cell Neurosci. 2017 Sep 5;11:262. doi: 10.3389/fncel.2017.00262. eCollection 2017.
Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell's stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 μM) or berbamine (≥1.55 μM) protect zebrafish hair cells from neomycin (6.25 μM, 1 h). Protection of zebrafish hair cells against gentamicin (10 μM, 6 h) was provided by ≥25 μM dTC or ≥12.5 μM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 μM, 48 h) by 20 μM berbamine or 25 μM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 μM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 μM (dTC) and 2.8 μM (berbamine) in the presence of 1.3 mM Ca at -104 mV. Berbamine, but not dTC, also blocks the hair cell's basolateral K current, I, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants.
氨基糖苷类抗生素被广泛用于治疗危及生命的细菌感染，但在相当一部分接受治疗的患者中会导致永久性听力丧失。这些抗生素通过位于毛细胞静纤毛顶端的机械电换能器（MET）通道进入内耳后，会对内耳的感觉毛细胞造成损伤。筒箭毒碱（dTC）是一种MET通道阻滞剂，可减少庆大霉素-德克萨斯红（GTTR）进入大鼠耳蜗毛细胞，并保护它们免受庆大霉素的损伤。小檗胺是一种结构相关的生物碱，可减少斑马鱼侧线毛细胞的GTTR标记，并保护它们免受氨基糖苷类诱导的细胞死亡。这两种化合物都被认为可减少氨基糖苷类通过MET通道进入毛细胞。在这里，我们表明dTC（≥6.25 μM）或小檗胺（≥1.55 μM）可保护斑马鱼毛细胞免受新霉素（6.25 μM，1小时）的损伤。≥25 μM的dTC或≥12.5 μM的小檗胺可保护斑马鱼毛细胞免受庆大霉素（10 μM，6小时）的损伤。在小鼠耳蜗培养物中，20 μM的小檗胺或25 μM的dTC可保护毛细胞免受长期暴露于庆大霉素（5 μM，48小时）的损伤。然而，小檗胺在较高浓度（≥30 μM）时对小鼠耳蜗毛细胞具有高毒性，而dTC则没有。斑马鱼试验中没有毒性提示在将斑马鱼神经丘的结果外推至哺乳动物耳蜗毛细胞时需谨慎。从小鼠外毛细胞（OHC）记录的MET电流表明，这两种化合物都是通透的开放通道阻滞剂，在-104 mV下1.3 mM Ca存在时，以2.2 μM（dTC）和2.8 μM（小檗胺）的半阻断浓度快速且可逆地阻断MET通道。小檗胺而非dTC还可阻断毛细胞的基底外侧钾电流I，模型研究表明小檗胺比dTC更容易透过MET通道。这些研究揭示了未来成功设计耳保护剂所需的MET通道阻滞剂的关键特性。
