Electrical activity encodes auditory and vestibular stimuli and sculpts peripheral and central nervous system pathways. The latter was demonstrated in the visual system, where spontaneous electrical activity regulates neural pathway formation. The underlying bases for electrical activity are pore forming proteins or ion channels inserted into the membrane of excitatory cells. In order to understand how this activity contributes to the normal and abnormal formation of the nervous system, one must answer the fundamental question of, what are the mechanisms that regulate ion channel expression during development? Potassium ion channels contribute to electrical activity in the inner ear by shaping the hair cell receptor potential and the signals transmitted to and from afferent and efferent nerve fibers, respectively. This activity can contribute to intracellular signals that regulate these cells during development. The long term goals of our research are to discover the intracellular pathways that regulate the expression of ion channels from gene to protein in the developing inner ear. These pathways are composed of protein-protein interactions that partner ion channels with transcription, cytoskeletal, chaperone, and clustering proteins, among others. Of the many K+ channels, the large conductance Ca2+-activated K+ or BK channel has a role not only in excitation, but also in intracellular signaling and metabolism. These functions are reflected in recent findings that show BK expresses, not only in the plasmalemma, but also in mitochondria and possibly the nuclear envelope. Given these different subcellular locations and the long, ligand-binding C-terminus of BK, we will examine BK interactions with BK-Associated Proteins (BKAPs) during early and late stages of mouse cochlear development. The proposed experiments focus on discovering and characterizing the role of BKAPs in promoting BK expression, function, and distribution by using proteomic and bioinformatic techniques.
The specific aims of the proposal are to (1) validate newly-discovered BKAPs from the adult cochlea, by measuring BK expression, when silencing its partners, (2) determine how specific BKAPs alter the biophysical properties of BK, (3) determine the properties of mitochondrial BK and its relation to specific BKAPs, and (4) map the BK interactome during early cochlear development. These studies will utilize proteomics, bioinformatics, siRNA, and electrophysiology.
These studies will begin to illuminate and characterize protein-protein interactions that guide normal ion channel development. Discovering these interactions is relevant to understanding the cochlear proteome and, with it, the many signals that contribute to the development and regeneration of the auditory and vestibular systems. Moreover, these discoveries will provide insights into major intracellular pathways that regulate not only normal cochlear development but those that lead to sensorineural deafness in children.
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