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      Functional Hair Cell Mechanotransducer Channels Are Required for Aminoglycoside Ototoxicity

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          Abstract

          Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.

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          Most cited references53

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          Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels.

          We describe a novel mechanism for vital fluorescent dye entry into sensory cells and neurons: permeation through ion channels. In addition to the slow conventional uptake of styryl dyes by endocytosis, small styryl dyes such as FM1-43 rapidly and specifically label hair cells in the inner ear by entering through open mechanotransduction channels. This labeling can be blocked by pharmacological or mechanical closing of the channels. This phenomenon is not limited to hair cell transduction channels, because human embryonic kidney 293T cells expressing the vanilloid receptor (TRPV1) or a purinergic receptor (P2X2) rapidly take up FM1-43 when those receptor channels are opened and not when they are pharmacologically blocked. This channel permeation mechanism can also be used to label many sensory cell types in vivo. A single subcutaneous injection of FM1-43 (3 mg/kg body weight) in mice brightly labels hair cells, Merkel cells, muscle spindles, taste buds, enteric neurons, and primary sensory neurons within the cranial and dorsal root ganglia, persisting for several weeks. The pattern of labeling is specific; nonsensory cells and neurons remain unlabeled. The labeling of the sensory neurons requires dye entry through the sensory terminal, consistent with permeation through the sensory channels. This suggests that organic cationic dyes are able to pass through a number of different sensory channels. The bright and specific labeling with styryl dyes provides a novel way to study sensory cells and neurons in vivo and in vitro, and it offers new opportunities for visually assaying sensory channel function.
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            FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel.

            Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.
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              The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels.

              The most serious side-effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular dihydrostreptomycin not only blocks the mechano-electrical transducer channels of mouse outer hair cells at negative membrane potentials, as previously shown, but also enters the cells through these channels, which are located in the cells' mechanosensory hair bundles. The voltage-dependent blocking kinetics indicate an open-channel block mechanism, which can be well described by a two barrier-one binding site model, quantifying the antibiotic's block of the channel as well as its permeation in terms of the associated rate constants. The results identify the open transducer channels as the main route for aminoglycoside entry. Intracellularly applied dihydrostreptomycin also blocks the transducer channels, but at positive membrane potentials. However, the potency of the block was two orders of magnitude lower than that due to extracellular dihydrostreptomycin. Extracellular Ca2+ increases the free energy of the barrier nearest the extracellular side and of the binding site for dihydrostreptomycin. This reduces both the entry of dihydrostreptomycin into the channel and the channel's affinity for the drug. In vivo, where the extracellular Ca2+ concentration in the endolymph surrounding the hair bundles is < 100 microM, we predict that some 9000 dihydrostreptomycin molecules per second enter each hair cell at therapeutic drug concentrations.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                26 July 2011
                : 6
                : 7
                : e22347
                Affiliations
                [1 ]Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
                [2 ]Department of Special Surgery, Mu'tah University, Alkarak, Jordan
                [3 ]Department of Otolaryngology-Head and Neck Surgery, Oregon Health & Science University, Portland, Oregon, United States of America
                [4 ]Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, United States of America
                Yale University, United States of America
                Author notes

                Conceived and designed the experiments: AJR AGC. Performed the experiments: AA LL MH AM AJR AGC. Analyzed the data: AA LL MH AJR AGC. Contributed reagents/materials/analysis tools: PSS AJR. Wrote the paper: AJR AGC.

                Article
                PONE-D-11-07057
                10.1371/journal.pone.0022347
                3144223
                21818312
                d6f8cd45-96ea-430c-b59b-dc93e8528796
                Alharazneh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 26 April 2011
                : 19 June 2011
                Page count
                Pages: 13
                Categories
                Research Article
                Biology
                Neuroscience
                Cellular Neuroscience
                Ion Channels
                Neurophysiology
                Peripheral Nervous System
                Sensory Systems
                Auditory System
                Neurobiology of Disease and Regeneration
                Toxicology
                Neurotoxicology
                Toxicokinetics

                Uncategorized
                Uncategorized

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