Abstract
Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca(2+)-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M. J., Dowdall, J. R., Khan, K. M., Hatfield, J. S., Ramakrishnan, N. A., and Drescher, D. G. (2012) Biochem. J. 443, 463-476). Here, we provide evidence for organ of Corti proteins, of Ca(2+)-dependent binding of the amino terminus of CNGA3 specifically to the carboxyl terminus of stereocilia tip-link protein CDH23 +68 (cadherin 23 with expressed exon 68) by yeast two-hybrid mating and co-transformation protocols, pulldown assays, and surface plasmon resonance analysis. Myosin VIIa, required for adaptation of hair cell mechanotransduction (MET) channel(s), competed with CDH23 +68, with direct Ca(2+)-dependent binding to the amino terminus of CNGA3. Based upon the premise that hair cell stereocilia tip-link proteins are closely coupled with MET, these results are consistent with the possibility that CNGA3 participates in hair-cell MET. Together with the demonstration of protein-protein interaction between HCN1 and tip-link protein protocadherin 15 CD3 (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227-3238; Ramakrishnan, N. A., Drescher, M. J., Khan, K. M., Hatfield, J. S., and Drescher, D. G. (2012) J. Biol. Chem. 287, 37628-37646), a protein-protein interaction for CNGA3 and a second tip-link protein, CDH23 +68, further suggests possible association of two different channels with a single stereocilia tip link.
Highlights
Stereocilia tip-link protein cadherin 23 couples mechanical forces to sensory transduction in inner ear receptor hair cells
The Amino Terminus of CNGA3 Binds to the Carboxyl Terminus of Cadherin 23 by Yeast Two-hybrid Analysis—Previously, a role for CNGA3 in vestibular hair cell function was implied from the finding of three forms of CNGA3 transcript expressed in a purified preparation of Ͼ106 hair cells [3]
Transcript and protein for cAMP-gated olfactory forms of Cyclic nucleotide-gated (CNG), namely CNGA2 and CNGA4, have been detected in mammalian cochlear hair cells [13], and CNGA3 transcript was detected in an organ of Corti cochlear subdissected fraction [14]
Summary
Stereocilia tip-link protein cadherin 23 couples mechanical forces to sensory transduction in inner ear receptor hair cells. The carboxyl terminus of highly Ca2؉-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, immunolocalizd to hair cell stereocilia We provide evidence for organ of Corti proteins, of Ca2؉-dependent binding of the amino terminus of CNGA3 to the carboxyl terminus of stereocilia tip-link protein CDH23 ؉68 (cadherin 23 with expressed exon 68) by yeast two-hybrid mating and co-transformation protocols, pulldown assays, and surface plasmon resonance analysis. We present biochemical evidence that the cytoplasmic amino terminus of ion channel CNGA3 binds the cytoplasmic carboxyl terminus of cadherin 23 (CDH23), a transmembrane protein that forms the upper two-thirds of filaments constituting hair cell stereocilia tip links. Myosin VIIa, a CDH23 binding partner [8], competes with CDH23 ϩ68 for interaction sites on CNGA3
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