Localization and expression of clarin-1, the Clrn1 gene product, in auditory hair cells and photoreceptors

Abstract

The Usher syndrome 3A (CLRN1) gene encodes clarin-1, which is a member of the tetraspanin family of transmembrane proteins. Although identified more than 6 years ago, little is known about its localization or function in the eye and ear. We developed a polyclonal antibody that react with all clarin-1 isoforms and used it to characterize protein expression in cochlea and retina. In the cochlea, we observe clarin-1expression in the stereocilia of P0 mice, and in synaptic terminals present at the base of the auditory hair cells from E18 to P6. In the retina, clarin-1 localizes to the connecting cilia, inner segment of photoreceptors and to the ribbon synapses. RT-PCR from P0 cochlea and P28 retina show mRNAs encoding only isoforms 2 and 3. Western-blots show that only isoform 2 is present in protein extracts from these same tissues. We examined clarin-1 expression in the immortomousederived hair cell line UB/OC-1. Only isoform 2 is expressed in UB/OC-1 at both mRNA and protein levels, suggesting this isoform is biologically relevant to hair cell function. The protein co-localizes with microtubules and post-transgolgi vesicles. The sub-cellular localization of clarin-1 in hair cells and photoreceptors suggests it functions at both the basal and apical poles of neurosensoriepithelia.

The role of primary cilia in the development and disease of the retina

“…115,229 The presence of clarin-1 (USH3A), which is mutated in Usher syndrome, further implicates the role of cilia proteins at the ribbon synapse in inherited disease. 230 …”

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

“…Amino acid alignments between clarin-1 and different members of the 4TM hyperfamily established a modest degree of similarity with stargazin, a calcium channel subunit involved in expression, mobilization, and clustering of AMPA (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors at the synaptic cleft. Since in the inner ear, clarin-1 is not only present at the apical and basal aspects of the hair cells but also in the afferent neurons that innervate them, a similar role in synaptic shaping has been suggested (Adato et al, 2002;Chen et al, 2003;Geng et al, 2009;Zallocchi et al, 2009).…”

Genetics of Nonsyndromic Recessively Inherited Moderate to Severe and Progressive Deafness in Humans

“…CLRN1 is a small 232 amino acid transmembrane protein which is present at both the basal and apical poles of the hair cells with expression being higher in outer hair cells as compared to the inner hair cells Zallocchi et al, 2009). Clrn1 is also expressed in the spiral ganglion Zallocchi et al, 2009).…”

“…Clrn1 is also expressed in the spiral ganglion Zallocchi et al, 2009). In mice, CLRN1 cannot be detected at the apex of hair cells at P1 and continues to be expressed in both the pre-and post-synaptic regions of outer hair cell type I afferent ribbon synapses (Zallocchi et al, 2009).…”

“…Clrn1 is also expressed in the spiral ganglion Zallocchi et al, 2009). In mice, CLRN1 cannot be detected at the apex of hair cells at P1 and continues to be expressed in both the pre-and post-synaptic regions of outer hair cell type I afferent ribbon synapses (Zallocchi et al, 2009). Additionally, CLRN1 may also have a role in actin assembly (Tian et al, 2009) and intracellular vesicle transport (Zallocchi et al, 2009).…”