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 expo sure 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 api cal surface. Cooling to 4 degreesC and high extracellular calcium both redu ce dye loading. Pretreatment with EGTA, a condition that breaks tip links a nd prevents mechanotransducer channel gating, abolishes subsequent dye load ing in the presence of calcium. Dye loading recovers after calcium chelatio n with a time course similar to that described for tip-link regeneration. M yo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundl es are stimulated by large excitatory stimuli. Extracellular perfusion of F M1-43 reversibly blocks mechano-transduction with half-blocking concentrati ons in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negat ive potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after v oltage steps to extreme potentials further suggests that FM1-43 competes wi th other cations for binding sites within the pore of the channel. FM1-43 d oes not block the transducer channel from the intracellular side at concent rations that would cause complete block when applied extracellularly. Calci um chelation and FM1-43 both reduce the ototoxic effects of the aminoglycos ide antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.