HCN1 and HCN2 proteins are expressed in cochlear hair cells: HCN1 can form a ternary complex with protocadherin 15 CD3 and F-actin-binding filamin A or can interact with HCN2.

A unique coupling between HCN1 and stereociliary tip-link protein protocadherin 15 has been described for a teleost vestibular hair-cell model and mammalian organ of Corti (OC) (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). We now show that Ca(2+)-dependent interaction of the organ of Corti HCN1 and protocadherin 15 CD3 is mediated by amino-terminal sequence specific to HCN1 and is not replicated by analogous specific peptides for HCN2 or HCN4 nor by amino-terminal sequence conserved across HCN isoforms utilized in channel formation. Furthermore, the HCN1-specific peptide binds both phosphatidylinositol (3,4,5)-trisphosphate and phosphatidylinositol (4,5)-bisphosphate but not phosphatidylinositol 4-phosphate. Singly isolated cochlear inner and outer hair cells express HCN1 transcript, and HCN1 and HCN2 protein is immunolocalized to hair-cell stereocilia by both z-stack confocal and pre-embedding EM immunogold microscopy, with stereociliary tip-link and subcuticular plate sites. indicates HCN1/HCN2/HCN3/HCN4 = 9:9:1:89 in OC of the wild-type mouse, with HCN4 protein primarily attributable to inner sulcus cells. A mutant form of HCN1 mRNA and protein is expressed in the OC of an HCN1 mutant, corresponding to a full-length sequence with the in-frame deletion of pore-S6 domains, predicted by construct. The mutant transcript of HCN1 is ∼9-fold elevated relative to wild-type levels, possibly representing molecular compensation, with unsubstantial changes in HCN2, HCN3, and HCN4. Immunoprecipitation protocols indicate alternate interactions of full-length proteins; HCN1 can interact with protocadherin 15 CD3 and F-actin-binding filamin A forming a complex that does not include HCN2, or HCN1 can interact with HCN2 forming a complex without protocadherin 15 CD3 but including F-actin-binding fascin-2.

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