Harmonin Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells

Department of Cell Biology, Institute for Childhood and Neglected Disease, The Scripps Research Institute, La Jolla, CA 92037, USA.
Neuron (Impact Factor: 15.05). 06/2009; 62(3):375-87. DOI: 10.1016/j.neuron.2009.04.006
Source: PubMed


In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement.

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    • "Mutations in this gene are also responsible for autosomal recessive non-syndromic deafness (Blaydon et al. 2003). USH1C encodes harmonin, which is a scaffolding protein that is required for the normal mechanosensory function of cochlear hair cells (Grillet et al. 2009). This protein might be involved in the maintenance of the synaptic structure or mediating the release of synaptic vesicles in the outer retina (Lillo et al. 2006). "
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    • "Calcium signal (DF/F) was calculated with the equation: (F-F 0 )/F 0 , where F 0 is the averaged fluorescence baseline at the beginning. Transduction current-displacement curves (I[X]) were fitted with a three state Boltzmann model to calculate channel open probability (P o ) as described (Grillet et al., 2009b). All data are mean ± SEM. "
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    ABSTRACT: Hair cells are mechanosensors for the perception of sound, acceleration, and fluid motion. Mechanotransduction channels in hair cells are gated by tip links, which connect the stereocilia of a hair cell in the direction of their mechanical sensitivity. The molecular constituents of the mechanotransduction channels of hair cells are not known. Here, we show that mechanotransduction is impaired in mice lacking the tetraspan TMHS. TMHS binds to the tip-link component PCDH15 and regulates tip-link assembly, a process that is disrupted by deafness-causing Tmhs mutations. TMHS also regulates transducer channel conductance and is required for fast channel adaptation. TMHS therefore resembles other ion channel regulatory subunits such as the transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory proteins (TARPs) of AMPA receptors that facilitate channel transport and regulate the properties of pore-forming channel subunits. We conclude that TMHS is an integral component of the hair cell's mechanotransduction machinery that functionally couples PCDH15 to the transduction channel.
    Cell 12/2012; 151(6):1283-95. DOI:10.1016/j.cell.2012.10.041 · 32.24 Impact Factor
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    • "We used the enhanced method to examine transfection of harmonin in hair cells. In mouse bundles harmonin-EGFP was located near stereocilia tips and was absent from tapers; this localization matched what is seen in acutely dissected mouse cochlea using anti-harmonin antibodies [3]. "
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    ABSTRACT: Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.
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