Structural and Molecular Basis of Transduction in Auditory Sensory Organs

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The hair cell mechanotransduction (MET) channel complex resides at the tips of the second and shorter rows of stereocilia in the hair bundle, which are both dynamic and prolate. In addition to TMC1 and TMC2, other transmembrane proteins have recently been identified as essential for normal MET. While TMC1 and TMC2 are candidates for the MET pore-forming channel proteins it is intriguing that they do not localize to stereocilia before P1-2 when other components of the MET apparatus is already in place. We hypothesized that there may be other TMCs that act as forerunners to TMC1 and TMC2 earlier in development. Reported proteomics and genomics data indicate that TMC4 and TMC5 are present in the organ of Corti. We generated knock in mice expressing both TMC4-GFP and TMC5-mCherry and made the striking discovery that TMC5 is present at tips of developing stereocilia in the organ of Corti between E17.5 and P2. After this point, TMC5 localization rapidly diminishes from stereocilia and TMC1 and TMC2 expression rises. After P2-3 TMC5 together with TMC4 is detected on the apical surface of supporting cells, and along and at the tip of their primary cilia. In vestibular hair cells we detected TMC4 and TMC5 in short, immature, hair bundles mostly at the periphery of the sensory epithelium. We also found that TMC4 and TMC5 interact with the MET proteins CIB2 and PCDH15 in a heterologous expression system. Together, these findings introduce TMC4 and TMC5 as potential precursors to TMC1 and TMC2 that likely play key roles in regulating stereocilia bundle development and/or function. Additionally, the function(s) of TMC4 and TMC5 in supporting cells and in their primary cilia remain to be elucidated. These findings provide new directions to investigate roles of TMCs in the formation of the MET complex and in other homeostatic functions in the developing organ of Corti. We are using single fluorophore counting methods to estimate the copy number of each of these fluorescently tagged proteins at the MET site to understand the compositional variation of the MET channel complex across stereocilia in a bundle, along the tonotopic gradient of the cochlea, and in different hair cell types. A putative variability in total number or stoichiometry of each of these proteins could provide insights into the extent of stochasticity in its assembly and regulation. Recent reports examining structural models of TMC1 based on the known structures of TMEM16 proteins, revealed the presence of a large cavity near the protein-lipid interface. This cavity harbors two residues that, when mutated, cause autosomal dominant hearing loss, suggesting that it could function as the elusive MET channel permeation pathway. To guide our experiments for probing the putative permeability properties of TMC4 and TMC5, we are collaborating with Angela Ballesteros (NINDS), to evaluate the potential structural and functional similarities between TMC1, 2, 4, and 5. Preliminary analyses indicate: 1) TMC4 and TMC5 share a common fold with TMC1, TMEM16, and TMEM63/OSCA proteins, consisting of 10 transmembrane helices and presenting a cavity that could function as a permeation pathway, 2) Differences in the amino acid composition of TMC1, 4 and 5 lead to changes in the electrostatic properties of the cavity; while the cavity of TMC1 is hydrophobic and anionic, the cavity in TMC5 is more cationic and in TMC4 is less charged. These differences in electrostatic properties of the cavity could grant ionic selectivity to the potential permeation pathways. We will be using early developing organ of Corti explants to test for permeability to aminoglycosides and 3kDa dextrans of different charges to evaluate the potential channel function of TMC4 and TMC5 in stereocilia and supporting cells. We have confirmed that TMC4 and TMC5 are also present in microvilli of intestinal epithelial cells and are using the intestinal epithelium as an accessible and robust model system to study TMC4 and TMC5 permeability, and for co-immunoprecipitation assays to identify candidate TMC binding partners. In collaboration with Jung-Bum Shin (UVA) we plan on examining the role of TMC4 and TMC5 in hair cells and supporting cells of the organ of Corti using TMC4 and TMC5 single and double KO mice. We previously showed that the appearance of the stereocilia cargo transporter MYO3A at stereocilia tips coincides with the onset of MET. MYO7A, another stereocilia myosin also shows a tip-to-base gradient of distribution consistent with tip-ward translocation and dynamic accumulation of this motor protein. Based on these observations, we hypothesize that MYO3A and MYO7A transport components of the MET complex to stereocilia tips with the potential for complementary function and/or redundancy. During the past year we refined our analyses of the ability of these two motor proteins to transport PCDH15 isoforms, which are components of the MET apparatus and involved in stereocilia bundle formation. Our new results show a cargo selectivity whereby MYO3A transports the PCDH15-CD2 isoform an integral component of the mature MET complex and MYO7A transports PCDH15-CD3 isoform previously localized at stereocilia tips in developing hair bundles. We also observed that in presence of the CD2 cargo there is a near two-fold increase in the enrichment of MYO3A at filopodia tips suggesting a cargo-dependent regulation of the myosin activity. In a collaboration with Jung-Bum Shin (UVA) and Anthony W. Peng (University of Colorado), we helped demonstrate that multiple MYO7A isoforms are expressed in the mouse cochlea. Two such isoforms MYO7A-C and MYO7A-S are generated by alternative transcription and translation start sites. MYO7A-C is expressed primarily in IHCs and in a tonotopic gradient in OHCs, with decreasing expression toward the cochlear base. These results are consistent with the observation of the expression levels of actin-GFP driven by a Myo7a-C promoter used in the Myo7a::Actin-GFP transgenic mouse. The Actin-GFP signal was predominantly observed in the IHCs and detected at low levels in the apical OHCs and decreased tonotopically toward the basal end of the cochlea. Myo7a-C mice show reduced levels of MYO7A at the UTLD and stereocilia base correlating with the observation that Myo7a-C hair cells show reduced MET resting channel open probability and slowed MET currents. Taken together, this collaborative study reveals unexpected isoform-specific differences in MYO7A expression in the cochlea and highlights the essential role of MYO7A in tensioning the hair cell MET complex. Spontaneous patterned action potential (AP) activity is considered important for the correct development of mammalian sensory systems. However, the relationship between Ca2+ AP activity in immature IHCs and the ATP-dependent intercellular Ca2+ signaling, which occurs spontaneously in cochlear non-sensory cells of the greater epithelial ridge, remains unclear. We performed Ca2+ imaging in organ of Corti explants from mice expressing genetically encoded calcium indicators and detected intrinsic spontaneous activity in IHCs. The independence of this spontaneous activity was corroborated by pharmacological experiments. These data have been combined with extensive imaging data using Ca2+ dyes and electrophysiology performed in the laboratories of Walter Marcotti (Uni. of Sheffield) and Fabio Mammano (Uni. of Padova) into a comprehensive study. Briefly, in this study we argue that a combination of intrinsic spontaneous Ca2+ activity and mutual influence between IHCs and non-sensory cells form an intricate feedback mechanism to control the level of AP synchronization in IHCs and generate the patterned activity implicated in the refinement of the auditory pathway before the onset of hearing.