The broad, long-range goal of this study is to determine the molecular mechanism of hearing and balance. Actin-filled projections, or stereocilia, on the sensory hair cells of the inner ear convert force produced by sounds and mechanical vibrations into nerve impulses. The prevailing model for mechanotransduction, thought to be well-conserved for both cochlear and vestibular hair cells, is one in which neighboring stereocilia connected by extracellular filaments called tip links, deflect in response to stimuli, thereby causing the opening or closing of transduction channels. The channels are attached to an adaptation-motor complex, which controls tip-link tension. During an excitatory stimulus, tension is initially high, opening channels; the motor complex then slips down the actin cytoskeleton, reducing tip-link tension and allowing the channels to close. By contrast, during a negative stimulus, tension is initially low and channels close; the motor complex then ascends the cytoskeleton, restoring the resting tension and reopening the channels. Localization of the molecular motor, myosin 1c (Myo1c) at strategic places in the stereocilia; and studies using mice expressing a mutant Myo1c that can be selectively inhibited have shown Myo1c's involvement in this process known as adaptation (Holt et al., 2002; Stauffer et al., 2005). Adaptation allows hair cells under prolonged stimuli to remain sensitive to new stimuli. The goal of this proposal is to determine how Myo1c supports specific aspects of adaptation by measuring adaptation in mice expressing Myo1c mutants with defined molecular properties. Myo1c mutants will include (i) those that affect the ability of Myo1c to adapt to mechanical load, a property predicted for Myo1c from previous studies in this laboratory (Batters et al., 2004a; 2004b); and (ii) those with aberrant sensitivity to Ca2+, which regulates Myo1c and adaptation.
The specific aims are: to express in vitro and characterize the biochemical and mechanical properties of Myo1c mutants using ATPase assays, motility assays, kinetic analyses and single-molecule mechanical studies. Next, knock-in mice expressing these mutant Myo1c molecules will be generated and adaptation will be measured in hair cells from the knock-in mice. The combined in vitro and animal studies are expected to provide critical, new insight into the molecular mechanism of Myo1c and its role in adaptation. This knowledge could ultimately lead to the design of rational diagnostics and therapies to treat diseases of hearing and/or balance. The proposed study focuses on the molecular motor protein, Myo1c, which is implicated as the adaptation-motor complex in the hair cells of the inner ear. Determining the biochemical and biophysical properties of key components of the transduction apparatus in hair cells, like Myo1c, and its role in adaptation are of fundamental importance to revealing the molecular mechanisms of hearing and balance, so that new and appropriate diagnostics and therapies to prevent or treat hearing loss and vertigo can be developed. ? ? ?
