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The Journal of Neuroscience, September 15, 2001, 21(18):7013-7025
FM1-43 Dye Behaves as a Permeant Blocker of the Hair-Cell Mechanotransducer Channel
J. E. Gale1, W. Marcotti1, 2, H. J. Kennedy2, C. J. Kros1, 2, and G. P. Richardson1 1 School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, United Kingdom, and 2 School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, United Kingdom
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°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.
Key words: hair cell; cochlea; mechanotransduction; ion channel; endocytosis; aminoglycosides; myosin VIIA; FM1-43
Copyright © 2001 Society for Neuroscience 0270-6474/01/21187013-13$05.00/0
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