The long-term objectives are to understand the cellular mechanisms of transduction in auditory hair cells and the factors underlying the tonotopic arrangement of these cells within the cochlea. The main focus will be on the role of intracellular Ca2+ in controlling transducer adaptation and hair bundle mechanics, through mechanics that are still imperfectly understood. Hair cell responses will be measured in the intact turtle cochlea and will be combined with intracellular Ca/2+ imaging.
Specific Aims are: (1) to determine the properties of the endogenous Ca/2+ buffering transducer adaptation in intact cells and in cells perfused with known Ca/2+ buffers; (2) to develop methods for recording single mechanotransducer channels both in intact hair cells and in isolated stereocilia, and to investigate their modulation by Ca/2+; (3) to determine the effects of Ca/2+ and Ca/2+ buffers on the mechanical properties of the hair bundles; (4) to study synaptic transmission between the hair cell and afferent nerve terminal by simultaneous recordings from the pre- and post-synaptic cells. Measurements of hair cell Ca/2+ will provide evidence about the Ca/2+ signals at the synaptic zones, and the contributions of mobile Ca/2+ buffers and mitochondria to Ca/2+ homeostasis. (5) to clone Ca/2+- activated K+ channels variants from turtle hair cells and characterize their kinetics and Ca2+ sensitivity by heterologous expression. Knowledge of the cochlear distribution of the K+ channel variants may lead to insights about how the tonotopic organization is established. The work seeks to elucidate the control of intracellular Ca/2+ and its mechanism of action at opposite poles of the hair cell: on the mechanotransducer channels, on the Ca/2+-activated K+ channels, and on the release of synaptic transmitter. Since hair cells experience large Ca2+ loads, disturbances of Ca/2+ balance-Ca/2+ excitotoxicity-may be the leading cause of hair cell damage. The mechanism by which internal Ca/2+ modulates the activity of the mechanotransducer channel is probably common to all hair cells, including those in the mammalian cochlea, and may be the site of irreversible damage during noise exposure, poisoning with ototoxic drugs or aging. Since the mechanotransducer channel may be the target of many diseases, acquired and genetic, exploring its mechanisms in situ could provide a rational approach to such clinical problems.
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