The Mechanosensory Structure of the Hair Cell Requires Clarin-1, a Protein Encoded by Usher Syndrome III Causative Gene

Otolaryngology Head and Neck Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 07/2012; 32(28):9485-98. DOI: 10.1523/JNEUROSCI.0311-12.2012
Source: PubMed


Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America.

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    • "Regarding FM1-43 uptake, a modest impairment has already been described for the Clrn1 / mice (Geng et al., 2012); however, this is the first study demonstrating an in vivo role for clarin-1 in mechanotransduction channel regulation and suggests that clarin-1 has similar functional roles at the apical domains of both fish and rodent hair cells. Given the almost complete blockage of FM1-43 uptake in the clarin-1 MOs and the observation of an interaction between Pchd15a and clarin-1, we were surprised to find that only a subpopulation of the hair cells showed apical expression of Pcdh15a. "
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    ABSTRACT: Clarin-1 is a four-transmembrane protein expressed by hair cells and photoreceptors. Mutations in its corresponding gene are associated with Usher syndrome type 3, characterized by late-onset and progressive hearing and vision loss in humans. Mice carrying mutations in the clarin-1 gene have hair bundle dysmorphology and a delay in synapse maturation. In this paper, we examined the expression and function of clarin-1 in zebrafish hair cells. We observed protein expression as early as 1 d postfertilization. Knockdown of clarin-1 resulted in inhibition of FM1-43 incorporation, shortening of the kinocilia, and mislocalization of ribeye b clusters. These phenotypes were fully prevented by co-injection with clarin-1 transcript, requiring its C-terminal tail. We also observed an in vivo interaction between clarin-1 and Pcdh15a. Altogether, our results suggest that clarin-1 is functionally important for mechanotransduction channel activity and for proper localization of synaptic components, establishing a critical role for clarin-1 at the apical and basal poles of hair cells.
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    • "ABR recordings were conducted as previously described [19]. Briefly, mice were anesthetized with dilute ketamine, xylazine, and acepromazine solution (15 mg ketamine, 3 mg xylazine, 0.5 mg acepromazine and sterile water/saline) I.P. alternating side of ejection if possible. "
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    PLoS ONE 11/2013; 8(11):e79226. DOI:10.1371/journal.pone.0079226 · 3.23 Impact Factor
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    • "To evaluate hearing, auditory-evoked brain stem response (ABRs) was recorded following presentation of pure tone stimulus to the mouse ear at frequencies of 8, 16 and 32 kHz in accordance with previous descriptions [20]. Briefly, mice were anesthetized with intraperitoneal injection of rodent cocktail (ketamine 40 mg/kg, xylazine 5 mg/kg, and acepromazine 1 mg/kg) at doses of 40, 5, and 1 mg/kg. "
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