Prestin, a motor protein serves as the cochlear amplifier contributing towards the sensitivity of the mammalian inner ear. Moreover, prestin expressed in heterologous systems yields functional characteristics that are not consistent with the bona-fide OHC properties, raising the possibility that there are missing prestin- interacting proteins that should be identified to understand the mechanisms of this remarkable motor protein. Using innovative genetic, molecular, cellular, and functional imaging strategies, we have invoke this hypothesis. In contrast to the dogma that prestin is uniquely OHC-specific, we hypothesize that the authentic cochlear amplifier consists of prestin and its family member, SLC26A6. We further hypothesize that SLC26A6 is required as an efficient transporter to create intracellular Cl- microdomain for the proper function of prestin. To test the hypothesis, we propose the following three aims: 1) To test the spatial expression and co- localization of prestin and slc26a6 in OHCs. First, we will determine the expression and co-localization of prestin and slc26a6 in OHCs using quantitative real-time PCR (qRT-PCR), immunofluorescence confocal microscopy and immuno-electron microscopy (immuno-EM). Prestin-/- and slc26a6-/- mice will serve as negative controls. 2) To test for the physical interaction or the hetero-oligomerization of prestin and slc26a6. To directly test for possible physical interaction or co-assembly between prestin and SLC26A6, we will use co- immunoprecipitation (co-IP) and fluorescence resonance energy transfer (FRET) techniques. 3) To determine the in vitro and in vivo functional roles of the interaction or hetero-oligomerization of prestin and slc26a6. We will record the anion exchange activities and the nonlinear capacitance of the co-expressed prestin and slc26a6. We will design prestin-slc26a6 chimera constructs to further test for function interaction of the two proteins. Finally, we will test the prediction that in the absence of slc26a6, the motor functions of prestin will be severely altered, compromising the hearing sensitivity of slc26a6 null mutant mice. These studies will reveal a missing functional component of the cochlear amplifier and may divulge a new therapeutic target for the treatment of hearing loss or deafness. The outcomes of these studies will alter our present understanding of the functional mechanisms of prestin in OHCs, and would shift the paradigm from ?prestin-centric? towards orchestral proteins that operate to amplify sound in the inner ear.
The mammalian inner ear has a remarkable ability to amplify sound through the activity of a motor protein called prestin. We will demonstrate that another protein, slc26a6, belonging to prestin's family members operate closely with prestin to confer sound amplification in the inner ear.
| Sirish, Padmini; Ledford, Hannah A; Timofeyev, Valeriy et al. (2017) Action Potential Shortening and Impairment of Cardiac Function by Ablation of Slc26a6. Circ Arrhythm Electrophysiol 10: |
