title: TRPA1-mediated accumulation of aminoglycosides in mouse cochlear outer hair cells.
abstract: 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.
authors: Department of Physiology, University of Kentucky, Lexington, KY 40536-0298, USA. Stepanyan@uky.edu
authors: Ruben S
authors: RS
authors: Stepanyan
authors: Artur A
authors: AA
authors: Indzhykulian
authors: A Catalina
authors: AC
authors: Vélez-Ortega
authors: Erich T
authors: ET
authors: Boger
authors: Peter S
authors: PS
authors: Steyger
authors: Thomas B
authors: TB
authors: Friedman
authors: Gregory I
authors: GI
authors: Frolenkov
mesh_terms: D000447:Aldehydes / Q000494:pharmacology; D000617:Aminoglycosides / Q000493:pharmacokinetics* / Q000633:toxicity*; D000818:Animals; D019556:COS Cells; D002412:Cations / Q000493:pharmacokinetics; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D005838:Genotype; D005839:Gentamicins / Q000493:pharmacokinetics* / Q000633:toxicity*; D057809:HEK293 Cells; D018072:Hair Cells, Auditory, Outer / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014966:Xanthenes / Q000493:pharmacokinetics
chemical_list: D000447:Aldehydes; D000617:Aminoglycosides; D002412:Cations; D015853:Cysteine Proteinase Inhibitors; C073804:FM1 43; D005456:Fluorescent Dyes; D005839:Gentamicins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels; C475685:Trpa1 protein, mouse; D014966:Xanthenes; C034657:Texas red; C027576:4-hydroxy-2-nonenal