PHARMACOLOGICAL ANALYSIS OF IONIC BALANCE IN HAIR CELLS

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The proper functioning of hair cells, the sensory transducing cells of the auditory and vestibular systems, depends on proper ionic compositions both outside and inside those cells. The nature of some insults to hair cells and of the resulting injuries suggest that pathophysiological changes to hair cells can involve loss of proper ionic balance (e.g., in temporary threshold shifts, acoustic trauma, ototoxicity, Meniere's disease, and aging). Loss of ionic balance would prevent normal hair cell function, and could lead to permanent damage, if for example, the hair cell consequently lose the ability to regulate cell volume. Since damage to hair cells is a frequent cause of deafness, understanding the ionic balance processes of hair cell may eventually help to prevent or minimize some forms of hearing loss. The long-term objective of this project is to determine how hair cells regulate their intracellular ionic state. This information will fill a major gap in knowledge about the normal physiology of hair cells and will eventually help to evaluate whether loss of ionic balance plays a role in pathophysiology. The experimental approach is to subject isolated hair cells to treatments designed to affect specific cellular mechanism that influence ion balance. Electron-probe microanalysis is then used to determine ion composition and net ion-transport rates of hair cells. This method allows quantitative stud of electrically silent transporters and active ion pumps that are hard to study with other techniques. Quantitative microfluorometry of ion-specific dyes will provide further information about intracellular binding or sequestration of ions once they have entered the hair cells. Cell volume will be measured with quantitative video microscopy. Changes in membrane potential will be determined. Work during the next project period will address fundamental questions about the individual ion-transport processes whose dynamic interactions establish ion balance in the cell. Experiments will examine how flows of sodium, potassium, chloride, calcium, an d hydrogen ion, through each of their major pathways across the hair-cell membrane, depend on ion concentration inside and outside the cell, on the energy sources that power ion transport, and on the membrane potential. The effects of these experiments on the intracellular handling of calcium and hydrogen ions will be determined. Since we will measure all the major permanent cell ions in these experiments, we will also be able to discover unexpected interactions among the individual ion-balance processes. In addition to their importance for understanding basic hair-cell physiology, these studies will forming a solid technical and intellectual basis for future work to evaluate the role of alterations of hair-cell ion balance in different forms of hearing loss.