Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells

^*Oregon Hearing Research Center and Vollum Institute, Portland, OR 97239; ^‡Department of Cytokine Biology, The Forsyth Institute, and Department of Developmental and Craniofacial Biology, Harvard Medical School, Boston, MA 02115; ^§Department of Otolaryngology, University of Tübingen, Elfriede-Aulhorn-Strasse 5, 72076 Tübingen, Germany; ^¶Department of Physiology II, University of Tübingen, Gmelinstrasse 5, 72076 Tübingen, Germany; ^††Max-Planck Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tübingen, Germany; and ^**Department of Physiology II, University of Würzburg, Röntgenring 9, 97070 Würzburg, Germany

Edited by Jeremy Nathans, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 19, 2005 (received for review March 23, 2005)

Abstract

In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.

Footnotes

↵ §§ To whom correspondence may be addressed. E-mail: gillespp{at}ohsu.edu or stefan.gruender{at}mail.uni-wuerzburg.de.
↵ † J.-B.S., D.A., and M.P. contributed equally to this work.
↵ ∥ Present address: Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.
↵ ‡‡ Present address: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Author contributions: M.L., P.G.G., and S.G. designed research; J.-B.S., D.A., M.P., M.S., M.L., and P.G.G. performed research; S.S. and S.G. contributed new reagents/analytic tools; M.L., P.G.G., and S.G. analyzed data; and P.G.G. and S.G. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TRP, transient receptor potential; Cdh23, cadherin 23; IFT, intraflagellar transport.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AJ576027).