The studies proposed here continue work on the molecular mechanism of sensory transduction by hair cells. A great deal of physiological evidence indicates that the transduction channels in vertebrate hair cells are directly activated by mechanical stimuli: that the tip links on stereocilia convey the displacement of the hair bundle to the channels, and that the stress causes channels to open. Similarly, adaptation of the transduction channels in the presence of a steady displacement is thought to come about by a decline in the stress reaching channels, by a movement of the tip link attachment. A particular myosin, type Ibeta, has been cloned from hair cells and is thought to be the motor protein that drives this movement. An additional phase of adaptation has been proposed to result from Ca++ binding directly to a site associated with the channel so as to close it. Both of these adaptation mechanisms can serve to regulate the set point for transduction. In this study, three aspects of the molecular apparatus mediating adaptation will be investigated. In the first part, the forces exerted by the hair bundle will be measured by the gradient-force light trap during adaptation. In this way, the contributions of a motor and a Ca++-dependent channel closure can be separated. In the second, the modulation of myosin-Ibeta by the second messenger cAMP will be studied. Myosin-Ibeta may be a substrate for protein kinase A, as part of a slower regulation of set point, and enzymes involved in cAMP metabolism may be part of the transduction apparatus. In the third, the yeast two-hybrid system will be used to find other proteins that bind to myosin- Ibeta, as a way of starting to identify all the proteins of the transduction complex. This understanding of hair bundle structures and the proteins associated with adaptation may elucidate certain pathological conditions of the auditory system. For instance, one of the myosins we cloned from hair cells is defective in Usher Syndrome, the most common inherited deafness in humans. Identification of the motor protein and proteins that bind to it may provide genetic tests for other inherited deafness.
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