Response units are indicated for binding with CD3-C at 61 mCa2+(red), 26

Response units are indicated for binding with CD3-C at 61 mCa2+(red), 26.5 mCa2+(green), or 1 mmEGTA (gray), compared with the response for bovine serum albumin (turquoise) or blank buffer (magenta).D, Ca2+dependence of the response (RU) for interaction of CD3-C (analyte) with HCN1-N (ligand). 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 Ca2+. In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by aKD= 2.39 10-7m. Ca2+at 26.5-68.0 mpromoted binding, withKD= 5.26 10-8m(at 61 mCa2+). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession numberXP_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 Ca2+-dependent, although the binding was skewed toward a lower effective maximum [Ca2+] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore existin vivobetween the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 mCa2+. Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells. HCN12is the primary full-length HCN isoform underlyingIh(hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation channel current) in a model hair cell preparation CACNB4 from the trout sacccule (1). cAMP-gatedIh, possibly in addition to the mechanosensory-transduction current, sets the membrane potential for a subpopulation of saccular hair cells (2,3). The membrane potential in the saccular hair cell subpopulation is sufficiently depolarized to activate voltage-gated calcium channels, permitting influx of calcium and secretion of hair cell transmitter (2). Given that saccular hair cells expressingIK1in addition toIhare more hyperpolarized, not supporting activation of the voltage-gated calcium channels, we predicted that spontaneous release of transmitter from the subpopulation of hair cells would constitute hair cell-generated spontaneous activity for the A2AR-agonist-1 saccule (1). However, little has been previously reported on the morphological localization of the HCN1 isoform in hair cells or possible links to structural proteins that mechanistically would localize HCN1 in hair cells (for preliminary report, see Ref.4). In general, little is known about protein-protein interactions for the HCN isoforms that would A2AR-agonist-1 modulateIhand/or the associated instantaneous current (5). Protocadherin 15 is a proposed tip link protein involved in connecting shorter stereocilia to adjacent taller stereocilia in the stereociliary array of inner ear hair cells, facilitating the opening of the mechanosensory transduction channel in response to auditory and vestibular stimuli. The active tip link protein inDanio reriois protocadherin 15a (6), characterized by splice variants in its carboxyl terminus. In the mammal, protocadherin 15 CD3 is hypothesized to be a tip link protein at insertion sites in the tips of the shorter stereocilia of the stereociliary array (7,8). == EXPERIMENTAL PROCEDURES == Acquisition of a Model Hair Cell Preparation from the Trout Saccule and an Organ of Corti Subfraction from the Rat CochleaHair cell layers were isolated from the trout saccule as previously described (9,10), yielding a hair cell preparation of 1 1.5 106saccular hair cells, free of intact supporting cells. An organ of Corti fraction was microdissected from rat cochlea (one-month-old ACI Black Agouti rats; Harlan Sprague-Dawley) as previously described and morphologically characterized (11). Yeast Two-hybrid Analysis: Trout Hair Cell LayerYeast two-hybrid screening for the interacting proteins of trout hair cell HCN1 was performed (Matchmaker kit; Clontech, Mountain View, CA) using the cytoplasmic amino terminus of trout saccular hair cell HCN1 (aa 1-118) as bait (seeFig. 1). PCR was carried out on trout hair cell cDNA with upstream primer 5-GCCGAATTCATGGAAGATAAATCAAATTCGTTC-3 and downstream primer 5-TGCGGATCCATAGGGATGAATGATCCA-3 (restriction sites underlined). The corresponding amplification product was purified, restriction-digested with EcoRI and BamHI enzymes and inserted into a similarly digested pGBKT7 vector to produce a fusion construct with the GAL4 DNA-binding domain. The fusion products were proven to be transcriptionally inactive in appropriate nutrient-deficient media, a necessary condition for unequivocal interpretation of results. == FIGURE 1. == Alignment (with OMIGA 2.0) of the amino terminus of rat organ of Corti HCN1 and trout saccular hair cell HCN1 (GenBank accession numberAAQ04053), both used as bait in yeast two-hybrid protocols, pull-down assays and SPR analyses.The cytoplasmic amino terminus sequence is encircled in a schematic depiction of HCN1 with S1-S6 A2AR-agonist-1 transmembrane regions and S4 (hatched) representing the voltage sensor. For rat organ of Corti HCN1, aa 11-18 display EF hand-like characteristics..