Gentamicin and FM1-43 enter sensory hair cells through mechanosensory transduction channel

Thesis (Ph. D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2002.Includes bibliographical references (p. 82-91).This dissertation describes a novel mechanism for the entry of organic cations, including the styryl pyridinium dyes and the notoriously ototo.xic aminoglycoside antibiotic drugs, into sensory hair cells: permeation through the transduction channels. Both gentamicin, an aminoglycoside, and FM1-43, a styryl pyridinium dye, rapidly and selectively enter hair cells. The hair cell transduction channel passes monovalent cations, but is selective for the divalent calcium. FM1-43 and gentamicin are larger than calcium, and FM1-43 is quite lipophilic. These experiments localize the rapid entry to the tips of the stereocilia, and show that perturbations of channel behavior also modulate the dye and drug entry, and that entry requires mechanical gating of the transduction channel. Channel expression in cultured cells also allows dye entry when those channels are opened. Less rapid routes also fill both hair cells and neighboring cells. Two lines of evidence extend these conclusions. First, voltage clamp recording confirmed gentamicin entry via the transduction channel: current was diminished but not blocked by the drug, and the block was not enhanced at more negative potentials. Second, injection of FM1-43 into mice labeled many sensory end organs and neurons. Limiting access-to the sensory endings diminished labeling. These results provide a powerful tool for visualizing the transduction process in living cells, for testing transduction and channel permeation, and perhaps for developing less toxic antibiotic drugs.by Richard B. MacDonald.Ph.D