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Localization of inner hair cell mechanotransducer channels using high-speed calcium imaging

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Abstract

Hair cells detect vibrations of their stereociliary bundle by activation of mechanically sensitive transducer channels. Although evidence suggests the transducer channels are near the stereociliary tops and are opened by force imparted by tip links connecting contiguous stereocilia, the exact channel site remains controversial. We used fast confocal imaging of fluorescence changes reflecting calcium entry during bundle stimulation to localize the channels. Calcium signals were visible in single stereocilia of rat cochlear inner hair cells and were up to tenfold larger and faster in the second and third stereociliary rows than in the tallest first row. The number of functional stereocilia was proportional to transducer current amplitude, indicating that there were about two channels per stereocilium. Comparable results were obtained in outer hair cells. The observations, supported by theoretical simulations, suggest there are no functional mechanically sensitive transducer channels in first row stereocilia and imply the channels are present only at the bottom of the tip links.

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Figure 1: Amplitude and time course of stereociliary calcium signals.
Figure 2: Amplitude and time course of stereociliary calcium signals of an inner hair cell bundle tilted to show all three stereociliary rows.
Figure 3: Variation in calcium signal along the stereocilia.
Figure 4: Relationship between the number of active stereocilia and mechanically sensitive transducer current amplitude.
Figure 5: Amplitude and time course of stereociliary calcium signals in an outer hair cell.
Figure 6: Theoretical distributions of calcium and Fluo-4FF fluorescence for two different channel locations.

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  • 12 April 2009

    In the version of this article initially published online, the name of the funding agency was spelt incorrectly in the acknowledgements section. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank Redshirt Imaging and Prairie Technologies for help in optimizing the confocal system. This work was supported by National Institute on Deafness and other Communication Disorders grants RO1 DC03896 to A.J.R. and DC01362 to R.F.

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Correspondence to Robert Fettiplace or Anthony J Ricci.

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Supplementary Figure 1 (PDF 207 kb)

Supplementary Video 1

Development of the calcium fluorescence in an inner hair cell bundle during hair bundle stimulation. The hair cell was depolarized to +100 mV (top trace, membrane potential) then the hair bundle was displaced (second trace, bundle displacement). The fluorescent signal began once the cell was repolarized to −80 mV, the signal being localized to the R2 stereocilia with no change in R1 stereocilia. (AVI 1572 kb)

Supplementary Video 2

Development of the calcium fluorescence in an outer hair cell bundle during hair bundle stimulation. The outer hair cell was depolarized to +100 mV (top trace, membrane potential) then the hair bundle displaced (second trace, bundle displacement). The fluorescent signal began once the cell was repolarized to −80 mV, the signal being localized to the R2 and R3 stereocilia with no change in R1 stereocilia. (AVI 1572 kb)

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Beurg, M., Fettiplace, R., Nam, JH. et al. Localization of inner hair cell mechanotransducer channels using high-speed calcium imaging. Nat Neurosci 12, 553–558 (2009). https://doi.org/10.1038/nn.2295

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