The least understood process of the inner ear, the transduction of mechanical stimuli into electrical responses, is also the stage at which severe pathology of the auditory and vestibular systems most often arises. We propose to employ the techniques of cell biology and membrane biophysics to investigate how transduction normally operates and how it is affected by a clinically important class of ototoxic agents, the aminoglycoside antibiotics. These studies will contribute to an understanding of transduction which, in the long term, will aid in the rational design of prophylaxis or treatment for conditions such as aminoglycoside ototoxicity, acoustic trauma, presbyacusis, and Meniere's disease. Hair cells from the bullfrog's inner ear will be maintained in vitro either within the saccular epithelium or as solitary cells dissociated by enzymes. The relationship between deflection of a cell's mechanosensitive hair bundle and the resultant receptor potential and membrane conductance changes will be carefully studied with stimuli of a variety of frequencies, amplitudes, and waveforms. The results will be of value in modeling the operation of less accessible organs such as the mammalian cochlea and vestibular apparatus. The subcellular site at which transduction occurs will be sought both by measuring the pattern of current flow around the hair bundle and by localizing the influx of calcium ion through activated transduction channels. The permeability of the transducer's ionic channel will be plumbed with radioactive tris(hydroxymethyl) aminomethane, an ion whose intracellular accumulation may also serve to label stimulated hair cells. As a step toward elucidation of the mechanism whereby aminoglycoside antibiotics damage hair cells, the site of action and binding kinetics of these drugs will be determined. It should be possible to learn whether ototoxic antibiotics interfere with cellular metabolism or exert their effects upon binding to a membrane receptor. Voltage-clamp experiments will be performed on isolated hair cells in order to estimate the conductance of individual transduction channels and to identify other membrane conductance mechanisms that affect the form of the receptor potential.
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