Fellowship for Isabel Aristizabal Ramirez: Molecular Mechanisms Regulating Activation of Mechano-Electrical Transducer Channels in Mammalian Auditory Hair Cells

In the inner ear, the vestibular system senses head position and movement whereas the cochlea detects sound. Although both organs use hair cells to convert mechanical stimuli into electrical signals, they have different frequency responses. Vestibular hair cells respond to relatively low frequencies, whereas mammalian cochlear hair cells detect sounds up to 100 kHz. It was once assumed that mechano-electrical transduction (MET) complexes are identical among vertebrate hair cells, but emerging evidence demonstrates significant differences in single channel properties and molecular composition of MET channels. Therefore, molecular variability between MET complexes may “tune” the hair cells for detection of particular frequencies. We and others have shown that CIB2 is an accessory subunit of the MET channel essential for hearing and mechanotransduction. The vestibular system contains CIB2 and CIB3 whereas cochlear hair cells only express CIB2. Interestingly, mutations in Cib2 that disrupt MET currents in auditory hair cells do not cause vestibular abnormalities. Only deletion of both, Cib2 and Cib3 results in vestibular deficits. These data along with previous studies showing that CIB3 partially restores MET currents in auditory hair cells lacking CIB2 suggest functional redundancy between CIB2 and CIB3. This raises the question of why cochlear hair cells express only CIB2. One possibility is that CIB2 may be required for detection of high frequency sound stimuli. The overall goal of this study is to explore the functional differences between CIB2 and CIB3 within MET complexes. In preliminary studies, I have designed a novel piezo-driven stiff probe that allows very fast deflection of the hair cell bundles and found that a deafness- related mutation in CIB2 results in slow activation of MET currents in the auditory hair cells. Therefore, I will perform whole-cell patch clamp recordings of MET currents in auditory hair cells of Cib2 knockout mice (that lack endogenous CIB2 and CIB3) after rescuing their mechanotransduction either with CIB2 or CIB3. The experiments will test the hypothesis that CIB2 confers faster MET channel activation in contrast to CIB3. Considering that mutations in CIB2 also cause mislocalization of membrane-associated proteins known to shape membrane curvature and stiffness, I will explore if CIB proteins influence MET complex kinetics by recruiting these membrane-associated proteins. Overall, this project will elucidate the functional differences between CIB2 and CIB3. Furthermore, this study is the first one that systematically explores the molecular mechanisms of MET channel activation in mammalian auditory hair cells, which was not possible before due to the lack of techniques for very fast hair bundle deflection.