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.
Gale
J E
JE
School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, United Kingdom.
Marcotti
W
W
Kennedy
H J
HJ
Kros
C J
CJ
Richardson
G P
GP
D000617:Aminoglycosides; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004398:Dyneins; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes / Q000493:pharmacokinetics / Q000494:pharmacology*; D006198:Hair Cells, Auditory / Q000187:drug effects* / Q000378:metabolism; D006579:Heterozygote; D006720:Homozygote; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D008465:Mechanoreceptors / Q000187:drug effects* / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008817:Mice, Mutant Strains; D000081412:Myosin VIIa; D009218:Myosins / Q000172:deficiency / Q000235:genetics; D018408:Patch-Clamp Techniques; D010812:Physical Stimulation; D011726:Pyridinium Compounds / Q000493:pharmacokinetics / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics / Q000494:pharmacology*; D013696:Temperature
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D002614:Chelating Agents; C073804:FM1 43; D005456:Fluorescent Dyes; D007473:Ion Channels; C000631456:Myo7a protein, mouse; D000081412:Myosin VIIa; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D004533:Egtazic Acid; D009218:Myosins; D004398:Dyneins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium