Abstract
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.
Highlights
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
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
Summary
Acquisition of a Model Hair Cell Preparation from the Trout Saccule and an Organ of Corti Subfraction from the Rat Cochlea—Hair cell layers were isolated from the trout saccule as previously described [9, 10], yielding a hair cell preparation of 1.5 ϫ 106 saccular hair cells, free of intact supporting. Immunofluorescence was examined with an Olympus BX60 microscope with fluorescein excitation (470 – 490 nm) and emission (500 – 800 nm) filters and photographed with a PM 20 camera system, and the negatives were digitized In this investigation of localization of HCN1 in the trout saccule and rat cochlea, two types of negative controls were performed; the primary antibodies were either preabsorbed with their respective antigens or omitted. Affinity-purified rat HCN1-N polypeptide (0.5 g) was mixed with bacterial lysate containing GST-CD3-C fusion protein (20 g) in PBST containing 1ϫ protease inhibitor mixture for 3 h at room temperature. PRSET-A fusion proteins were made from rat organ of Corti cDNA for HCN1-N (see previous section for GST pulldown assays), the carboxyl terminus of protocadherin 15 CD3 (CD3-C) and its deletion mutants. The determination of Ca2ϩ dependence of binding was based upon the anal- RESULTS
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