Calcium-dependent Binding of HCN1 Channel Protein to Hair Cell Stereociliary Tip Link Protein Protocadherin 15 CD3

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 HCN12 is the primary full-length HCN isoform underlying I h (hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation channel current) in a model hair cell preparation from the trout sacccule (1). cAMP-gated I h , 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 expressing I K1 in addition to I h are 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 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 proteinprotein interactions for the HCN isoforms that would modulate I h and/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 in Danio rerio is 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 theRat Cochlea-Hair cell layers were isolated from the t...

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

“…2C), 2-month-old CBA J mice of either sex (The Jackson Laboratories, Bar Harbor, ME) were the source of bony cochleas fixed in 4% formaldehyde, 0.1% glutaraldehyde and decalcified with protocols described previously (14). Deparaffinized sections (4 -5 m) were incubated overnight at 4°C with primary antibody (the custom polyclonal rabbit antibody targeting rat otoferlin diluted 1:1,500 in PBS containing chick serum).…”

Cyclic Nucleotide-gated Channel α-3 (CNGA3) Interacts with Stereocilia Tip-Link Cadherin 23 + Exon 68 or Alternatively with Myosin VIIa, Two Proteins Required for Hair Cell Mechanotransduction

“…Ehrlicher et al (54) determined that mechanical strain in actin networks directly differentially regulates protein-protein interactions of filamin A. A correlate to this finding would be the existence of alternate protein complexes, a model that we have confirmed for proteins expressed in hair cells (10,12). Filamin A, in immunoprecipitations of full-length proteins, binds HCN1 and tip link-protein protocadherin 15 CD3, but this protein complex does not include HCN2.…”

“…PCDH15, constituting the lower 1/3 of the tip link, is inserted via its cytoplasmic carboxyl terminus at the top of the shorter stereocilium (24). CNGA3 and HCN1, representing stereociliary proteins (3,10,12), interact with CDH23 ϩ68 and PCDH15 CD3, respectively. In one configuration, as a consequence of specific Ca 2ϩ -dependent molecular interactions, HCN1 is localized to the top of the shorter stereocilium and CNGA3 to a lateral position on the next taller stereocilium.…”

Preliminary studies on differential expression of auditory functional genes in the brain after repeated blast exposures

“…Cadherin and protocadherin mutations are reported to be involved in noise-induced hearing loss [13][14][16][17]44]. The altered expression profile of cadherins and protocadherins in different regions of the brain after repeated blast exposures may impair central auditory processing.…”