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

Abstract

Mutation in the clarin-1 gene 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 (Clrn1−/−) gene 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 Ca2+ currents and membrane capacitance from IHCs that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 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 (Clrn1N48K) supports our in vitro and Clrn1−/− mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Further, the ear phenotype in the Clrn1N48K mouse suggests that it is a valuable model for ear disease in CLRN1N48K, the most prevalent Usher III mutation in North America.

Clarin-1 acts as a modulator of mechanotransduction activity and presynaptic ribbon assembly

“…This result contrasts with the published data in the Clrn1 / mice (Geng et al, , 2012 in which the impairment in dye uptake and the few tip links correlate with hair cell bundle abnormalities. Although speculative, these discrepancies may be explained by a compensatory mechanism through the zebrafish pcdh15a orthologue, pcdh15b.…”

“…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.…”

“…As for clarin-1, this is the first work presenting evidence that suggests a direct role in ciliogenesis for an Usher protein. Because Usher mouse models show a disruption of the hair cell bundle integrity (Lefèvre et al, 2008), most of the studies related to clarin-1 at the apical aspect of hair cells have been focused on the stereocilia Geng et al, 2009Geng et al, , 2012. Its putative role in kinocilium formation, before this work, was unexplored.…”

“…These studies have shown that, similar to other Usher proteins, clarin-1 is necessary for hair bundle and ribbon synapse development and maintenance. Studies in the clarin-1 mutant mouse (Clrn1 / ) showed variable vestibular dysfunction, progressive hearing loss, compromised hair bundle integrity, and a delay in ribbon synapse maturation and neuronal activation (Geng et al, , 2012Zallocchi et al, 2012b). Expression of the clarin-1 orthologue has also been demonstrated in zebrafish, in which it may be functioning as a scaffold protein between different retinal cell types .…”

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome

“…CLRN1 isoform 2 encodes a 232-amino acid tetraspan-like glycoprotein (1,4,6). Studies of mutant mice lacking exon 1 of Clrn1 (7,8) and knockdown approaches in zebrafish (9,10) have revealed structural and functional defects of F-actin-enriched stereocilia in hair cells. Additional roles for clarin-1 have also been sought, as mouse Clrn1 transcripts have been detected in both hair cells and the auditory primary neurons, from embryonic day 16 (E16) to adult stages (1,11).…”