Calcium-dependent binding of HCN1 channel protein to hair cell stereociliary tip link protein protocadherin 15 CD3.

The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca(2+). In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K(D) = 2.39 x 10(-7) m. Ca(2+) at 26.5-68.0 microm promoted binding, with K(D) = 5.26 x 10(-8) m (at 61 microm Ca(2+)). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession number XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca(2+)-dependent, although the binding was skewed toward a lower effective maximum [Ca(2+)] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 microm Ca(2+). Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells.

Expression of subunits for the cAMP-sensitive 'olfactory' cyclic nucleotide-gated ion channel in the cochlea: implications for signal transduction.

Cyclic nucleotide-gated (CNG) ion channels have been implicated as functioning in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. The olfactory, cAMP-sensitive CNG ion channel in vivo is considered to comprise the pore-forming CNG2 subunit together with CNG5 and CNG4.3 modulatory subunits. The expression of these 'olfactory' CNG subunit transcripts in microdissected subfractions of the rat cochlea and hair cell libraries has been investigated with RT-PCR. Unmodified transcripts of CNG2 were detected in the organ of Corti, lateral wall and spiral ganglion subfractions. CNG5 message was found in both the sensory organ of Corti and the non-sensory lateral wall subfractions but not in the spiral ganglion subfraction. The CNG5 sequence obtained for the organ of Corti fraction encompassed 78% of the olfactory CNG5 cDNA sequence. CNG5 message has also been detected in an inner hair cell cDNA library. In the lateral wall, unmodified CNG5 sequence was observed as well as truncated versions of CNG5 transcripts, one of which was also found in the rat brain. The truncated versions were characterized by deletions that resulted in a shift in reading frame and the premature appearance of a stop codon. The 'olfactory' CNG4.3 cDNA was amplified from all three subfractions. Within the cochlea, CNG2 immunoreactivity was selectively distributed in a pattern similar to that of adenylyl cyclase type I. Immunoreactivity to CNG2 has been localized to stereocilia of inner hair cells. CNG5 immunoreactivity was associated with stereocilia and lateral plasma membranes of outer hair cells. We conclude that transcripts necessary for a functional cAMP-sensitive CNG ion channel are present in the cochlea resulting from combinations of CNG2 with CNG5 and CNG4.3. Further, the localization of CNG2 and CNG5 immunoreactivity to hair cell stereocilia suggests a role for cAMP-sensitive CNG channels in hair cell signal transduction.