Molecular and cytological profiling of biological aging of mouse cochlear inner and outer hair cells.

Age-related hearing loss (ARHL) negatively impacts quality of life in the elderly population. The prevalent cause of ARHL is loss of mechanosensitive cochlear hair cells (HCs). The molecular and cellular mechanisms of HC degeneration remain poorly understood. Using RNA-seq transcriptomic analyses of inner and outer HCs isolated from young and aged mice, we show that HC aging is associated with changes in key molecular processes, including transcription, DNA damage, autophagy, and oxidative stress, as well as genes related to HC specialization. At the cellular level, HC aging is characterized by loss of stereocilia, shrinkage of HC soma, and reduction in outer HC mechanical properties, suggesting that functional decline in mechanotransduction and cochlear amplification precedes HC loss and contributes to ARHL. Our study reveals molecular and cytological profiles of aging HCs and identifies genes such as Sod1, Sirt6, Jund, and Cbx3 as biomarkers and potential therapeutic targets for ameliorating ARHL.

Interaction with ectopic cochlear crista sensory epithelium disrupts basal cochlear sensory epithelium development in Lmx1a mutant mice.

The LIM homeodomain transcription factor Lmx1a shows a dynamic expression in the developing mouse ear that stabilizes in the non-sensory epithelium. Previous work showed that Lmx1a functional null mutants have an additional sensory hair cell patch in the posterior wall of a cochlear duct and have a mix of vestibular and cochlear hair cells in the basal cochlear sensory epithelium. In E13.5 mutants, Sox2-expressing posterior canal crista is continuous with an ectopic "crista sensory epithelium" located in the outer spiral sulcus of the basal cochlear duct. The medial margin of cochlear crista is in contact with the adjacent Sox2-expressing basal cochlear sensory epithelium. By E17.5, this contact has been interrupted by the formation of an intervening non-sensory epithelium, and Atoh1 is expressed in the hair cells of both the cochlear crista and the basal cochlear sensory epithelium. Where cochlear crista was formerly associated with the basal cochlear sensory epithelium, the basal cochlear sensory epithelium lacks an outer hair cell band, and gaps are present in its associated Bmp4 expression. Further apically, where cochlear crista was never present, the cochlear sensory epithelium forms a poorly ordered but complete organ of Corti. We propose that the core prosensory posterior crista is enlarged in the mutant when the absence of Lmx1a expression allows JAG1-NOTCH signaling to propagate into the adjacent epithelium and down the posterior wall of the cochlear duct. We suggest that the cochlear crista propagates in the mutant outer spiral sulcus because it expresses Lmo4 in the absence of Lmx1a.

Transcriptomes of cochlear inner and outer hair cells from adult mice.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two anatomically and functionally distinct types of mechanosensitive receptor cells in the mammalian cochlea. The molecular mechanisms defining their morphological and functional specializations are largely unclear. As a first step to uncover the underlying mechanisms, we examined the transcriptomes of IHCs and OHCs isolated from adult CBA/J mouse cochleae. One thousand IHCs and OHCs were separately collected using the suction pipette technique. RNA sequencing of IHCs and OHCs was performed and their transcriptomes were analyzed. The results were validated by comparing some IHC and OHC preferentially expressed genes between present study and published microarray-based data as well as by real-time qPCR. Antibody-based immunocytochemistry was used to validate preferential expression of SLC7A14 and DNM3 in IHCs and OHCs. These data are expected to serve as a highly valuable resource for unraveling the molecular mechanisms underlying different biological properties of IHCs and OHCs as well as to provide a road map for future characterization of genes expressed in IHCs and OHCs.

Cell-Specific Transcriptome Analysis Shows That Adult Pillar and Deiters' Cells Express Genes Encoding Machinery for Specializations of Cochlear Hair Cells.

The mammalian auditory sensory epithelium, the organ of Corti, is composed of hair cells and supporting cells. Hair cells contain specializations in the apical, basolateral and synaptic membranes. These specializations mediate mechanotransduction, electrical and mechanical activities and synaptic transmission. Supporting cells maintain homeostasis of the ionic and chemical environment of the cochlea and contribute to the stiffness of the cochlear partition. While spontaneous proliferation and transdifferentiation of supporting cells are the source of the regenerative response to replace lost hair cells in lower vertebrates, supporting cells in adult mammals no longer retain that capability. An important first step to revealing the basic biological properties of supporting cells is to characterize their cell-type specific transcriptomes. Using RNA-seq, we examined the transcriptomes of 1,000 pillar and 1,000 Deiters' cells, as well as the two types of hair cells, individually collected from adult CBA/J mouse cochleae using a suction pipette technique. Our goal was to determine whether pillar and Deiters' cells, the commonly targeted cells for hair cell replacement, express the genes known for encoding machinery for hair cell specializations in the apical, basolateral, and synaptic membranes. We showed that both pillar and Deiters' cells express these genes, with pillar cells being more similar to hair cells than Deiters' cells. The fact that adult pillar and Deiters' cells express the genes cognate to hair cell specializations provides a strong molecular basis for targeting these cells for mammalian hair cell replacement after hair cells are lost due to damage.

Characterization of Hair Cell-Like Cells Converted From Supporting Cells After Notch Inhibition in Cultures of the Organ of Corti From Neonatal Gerbils.

The senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Hair cells transduce mechanical stimuli into electrical activity. Loss of hair cells as a result of aging or exposure to noise and ototoxic drugs is the major cause of noncongenital hearing and balance deficits. In the ear of non-mammals, lost hair cells can spontaneously be replaced by production of new hair cells from conversion of supporting cells. Although supporting cells in adult mammals have lost that capability, neonatal supporting cells are able to convert to hair cells after inhibition of Notch signaling. We questioned whether Notch inhibition is sufficient to convert supporting cells to functional hair cells using electrophysiology and electron microscopy. We showed that pharmacological inhibition of the canonical Notch pathway in the cultured organ of Corti prepared from neonatal gerbils induced stereocilia formation in supporting cells (defined as hair cell-like cells or HCLCs) and supernumerary stereocilia in hair cells. The newly emerged stereocilia bundles of HCLCs were functional, i.e., able to respond to mechanical stimulation with mechanotransduction (MET) current. Transmission electron microscopy (TEM) showed that HCLCs converted from pillar cells maintained the pillar cell shape and that subsurface cisternae, normally observed underneath the cytoskeleton in outer hair cells (OHCs), was not present in Deiters' cells-derived HCLCs. Voltage-clamp recordings showed that whole-cell currents from Deiters' cells-derived HCLCs retained the same kinetics and magnitude seen in normal Deiters' cells and that nonlinear capacitance (NLC), an electrical hallmark of OHC electromotility, was not detected from any HCLCs measured. Taken together, these results suggest that while Notch inhibition is sufficient for promoting stereocilia bundle formation, it is insufficient to convert neonatal supporting cells to mature hair cells. The fact that Notch inhibition led to stereocilia formation in supporting cells and supernumerary stereocilia in existing hair cells appears to suggest that Notch signaling may regulate stereocilia formation and stability during development.

RNA-seq transcriptomic analysis of adult zebrafish inner ear hair cells.

Although hair cells are the sensory receptors of the auditory and vestibular systems in the ears of all vertebrates, hair cell properties are different between non-mammalian vertebrates and mammals. To understand the basic biological properties of hair cells from non-mammalian vertebrates, we examined the transcriptome of adult zebrafish auditory and vestibular hair cells. GFP-labeled hair cells were isolated from inner-ear sensory epithelia of a pou4f3 promoter-driven GAP-GFP line of transgenic zebrafish. One thousand hair cells and 1,000 non-sensory surrounding cells (nsSCs) were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates for the two cell types was performed and analyzed. Comparisons between hair cells and nsSCs allow identification of enriched genes in hair cells, which may underlie hair cell specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult zebrafish hair cells. It also establishes a framework for future characterization of genes expressed in hair cells and the study of hair cell evolution.

Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells.

Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.

Transcription Factors Expressed in Mouse Cochlear Inner and Outer Hair Cells.

Regulation of gene expression is essential to determining the functional complexity and morphological diversity seen among different cells. Transcriptional regulation is a crucial step in gene expression regulation because the genetic information is directly read from DNA by sequence-specific transcription factors (TFs). Although several mouse TF databases created from genome sequences and transcriptomes are available, a cell type-specific TF database from any normal cell populations is still lacking. We identify cell type-specific TF genes expressed in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) using hair cell-specific transcriptomes from adult mice. IHCs and OHCs are the two types of sensory receptor cells in the mammalian cochlea. We show that 1,563 and 1,616 TF genes are respectively expressed in IHCs and OHCs among 2,230 putative mouse TF genes. While 1,536 are commonly expressed in both populations, 73 genes are differentially expressed (with at least a twofold difference) in IHCs and 13 are differentially expressed in OHCs. Our datasets represent the first cell type-specific TF databases for two populations of sensory receptor cells and are key informational resources for understanding the molecular mechanism underlying the biological properties and phenotypical differences of these cells.

Glutamate transporter homolog-based model predicts that anion-π interaction is the mechanism for the voltage-dependent response of prestin.

Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid, and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions are still unknown. Our three-dimensional model of prestin using molecular dynamics simulations predicts that prestin contains eight transmembrane-spanning segments and two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Tyr(367), Tyr(486), Tyr(501), and Tyr(508) contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters, and motor proteins.

Characterization of transcriptomes of cochlear inner and outer hair cells.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two types of sensory receptor cells that are critical for hearing in the mammalian cochlea. IHCs and OHCs have different morphology and function. The genetic mechanisms that define their morphological and functional specializations are essentially unknown. The transcriptome reflects the genes that are being actively expressed in a cell and holds the key to understanding the molecular mechanisms of the biological properties of the cell. Using DNA microarray, we examined the transcriptome of 2000 individually collected IHCs and OHCs from adult mouse cochleae. We show that 16,647 and 17,711 transcripts are expressed in IHCs and OHCs, respectively. Of those genes, ∼73% are known genes, 22% are uncharacterized sequences, and 5.0% are noncoding RNAs in both populations. A total of 16,117 transcripts are expressed in both populations. Uniquely and differentially expressed genes account for <15% of all genes in either cell type. The top 10 differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, and Map4k4 in IHCs and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, and Ankrd22 in OHCs. We analyzed commonly and differentially expressed genes with the focus on genes related to hair cell specializations in the apical, basolateral, and synaptic membranes. Eighty-three percent of the known deafness-related genes are expressed in hair cells. We also analyzed genes involved in cell-cycle regulation. Our dataset holds an extraordinary trove of information about the molecular mechanisms underlying hair cell morphology, function, pathology, and cell-cycle control.

Prestin at year 14: progress and prospect.

Prestin, the motor protein of cochlear outer hair cells, was identified 14 years ago. Prestin-based outer hair cell motility is responsible for the exquisite sensitivity and frequency selectivity seen in the mammalian cochlea. Prestin is the 5th member of an eleven-member membrane transporter superfamily of SLC26A proteins. Unlike its paralogs, which are capable of transporting anions across the cell membrane, prestin primarily functions as a motor protein with unique capability of performing direct and reciprocal electromechanical conversion on microsecond time scale. Significant progress in the understanding of its structure and the molecular mechanism has been made in recent years using electrophysiological, biochemical, comparative genomics, structural bioinformatics, molecular dynamics simulation, site-directed mutagenesis and domain-swapping techniques. This article reviews recent advances of the structural and functional properties of prestin with focus on the areas that are critical but still controversial in understanding the molecular mechanism of how prestin works: The structural domains for voltage sensing and interaction with anions and for conformational change. Future research directions and potential application of prestin are also discussed. This article is part of a Special Issue entitled .

Lizard and frog prestin: evolutionary insight into functional changes.

The plasma membrane of mammalian cochlear outer hair cells contains prestin, a unique motor protein. Prestin is the fifth member of the solute carrier protein 26A family. Orthologs of prestin are also found in the ear of non-mammalian vertebrates such as zebrafish and chicken. However, these orthologs are electrogenic anion exchangers/transporters with no motor function. Amphibian and reptilian lineages represent phylogenic branches in the evolution of tetrapods and subsequent amniotes. Comparison of the peptide sequences and functional properties of these prestin orthologs offer new insights into prestin evolution. With the recent availability of the lizard and frog genome sequences, we examined amino acid sequence and function of lizard and frog prestins to determine how they are functionally and structurally different from prestins of mammals and other non-mammals. Somatic motility, voltage-dependent nonlinear capacitance (NLC), the two hallmarks of prestin function, and transport capability were measured in transfected human embryonic kidney cells using voltage-clamp and radioisotope techniques. We demonstrated that while the transport capability of lizard and frog prestin was compatible to that of chicken prestin, the NLC of lizard prestin was more robust than that of chicken's and was close to that of platypus. However, unlike platypus prestin which has acquired motor capability, lizard or frog prestin did not demonstrate motor capability. Lizard and frog prestins do not possess the same 11-amino-acid motif that is likely the structural adaptation for motor function in mammals. Thus, lizard and frog prestins appear to be functionally more advanced than that of chicken prestin, although motor capability is not yet acquired.

Proteomic analysis of the organ of corti using nanoscale liquid chromatography coupled with tandem mass spectrometry.

The organ of Corti (OC) in the cochlea plays an essential role in auditory signal transduction in the inner ear. For its minute size and trace amount of proteins, the identification of the molecules in pathophysiologic processes in the bone-encapsulated OC requires both delicate separation and a highly sensitive analytical tool. Previously, we reported the development of a high resolution metal-free nanoscale liquid chromatography system for highly sensitive phosphoproteomic analysis. Here this system was coupled with a LTQ-Orbitrap XL mass spectrometer to investigate the OC proteome from normal hearing FVB/N male mice. A total of 628 proteins were identified from six replicates of single LC-MS/MS analysis, with a false discovery rate of 1% using the decoy database approach by the OMSSA search engine. This is currently the largest proteome dataset for the OC. A total of 11 proteins, including cochlin, myosin VI, and myosin IX, were identified that when defective are associated with hearing impairment or loss. This study demonstrated the effectiveness of our nanoLC-MS/MS platform for sensitive identification of hearing loss-associated proteins from minute amount of tissue samples.

A motif of eleven amino acids is a structural adaptation that facilitates motor capability of eutherian prestin.

Cochlear outer hair cells (OHCs) alter their length in response to transmembrane voltage changes. This so-called electromotility is the result of conformational changes of membrane-bound prestin. Prestin-based OHC motility is thought to be responsible for cochlear amplification, which contributes to the exquisite frequency selectivity and sensitivity of mammalian hearing. Prestin belongs to an anion transporter family, the solute carrier protein 26A (SLC26A). Prestin is unique in this family in that it functions as a voltage-dependent motor protein manifested by two hallmarks, nonlinear capacitance and motility. Evidence suggests that prestin orthologs from zebrafish and chicken are anion exchangers or transporters with no motor function. We identified a segment of 11 amino acid residues in eutherian prestin that is extremely conserved among eutherian species but highly variable among non-mammalian orthologs and SLC26A paralogs. To determine whether this sequence represents a motif that facilitates motor function in eutherian prestin, we utilized a chimeric approach by swapping corresponding residues from the zebrafish and chicken with those of gerbil. Motility and nonlinear capacitance were measured from chimeric prestin-transfected human embryonic kidney 293 cells using a voltage-clamp technique and photodiode-based displacement measurement system. We observed a gain of motor function with both of the hallmarks in the chimeric prestin without loss of transport function. Our results show, for the first time, that the substitution of a span of 11 amino acid residues confers the electrogenic anion transporters of zebrafish and chicken prestins with motor-like function. Thus, this motif represents the structural adaptation that assists gain of motor function in eutherian prestin.

Expression of Neurog1 instead of Atoh1 can partially rescue organ of Corti cell survival.

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1(KINeurog1)) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1(+/KINeurog1)) and homozygous (Atoh1(KINeurog1/KINeurog1)) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete 'flat epithelium' in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1(f/+)), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1(KINeurog1) can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons.

Engineered pendrin protein, an anion transporter and molecular motor.

Pendrin and prestin both belong to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Pendrin (SLC26A4) is a chloride-iodide transporter that is found at the luminal membrane of follicular cells in the thyroid gland as well as in the endolymphatic duct and sac of the inner ear, whereas prestin (SLC26A5) is expressed in the plasma membrane of cochlear outer hair cells and functions as a unique voltage-dependent motor. We recently identified a motif that is critical for the motor function of prestin. We questioned whether it was possible to create a chimeric pendrin protein with motor capability by integrating this motility motif from prestin. The chimeric pendrin was constructed by substituting residues 160-179 in human pendrin with residues 156-169 from gerbil prestin. Non-linear capacitance and somatic motility, two hallmarks representing prestin function, were measured from chimeric pendrin-transfected human embryonic kidney 293 cells using the voltage clamp technique and photodiode-based displacement measurement system. We showed that this 14-amino acid substitution from prestin was able to confer pendrin with voltage-dependent motor capability despite the amino acid sequence disparity between pendrin and prestin. The molecular mechanism that facilitates motor function appeared to be the same as prestin because the motor activity depended on the concentration of intracellular chloride and was blocked by salicylate treatment. Radioisotope-labeled formate uptake measurements showed that the chimeric pendrin protein retained the capability to transport formate, suggesting that the gain of motor function was not at the expense of its inherent transport capability. Thus, the engineered pendrin was capable of both transporting anions and generating force.

MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival.

MicroRNAs (miRNAs) post-transcriptionally repress complementary target gene expression and can contribute to cell differentiation. The coordinate expression of miRNA-183 family members (miR-183, miR-96, and miR-182) has been demonstrated in sensory cells of the mouse inner ear and other vertebrate sensory organs. To further examine hair cell miRNA expression in the mouse inner ear, we have analyzed miR-183 family expression in wild type animals and various mutants with defects in neurosensory development. miR-183 family member expression follows neurosensory cell specification, exhibits longitudinal (basal-apical) gradients in maturating cochlear hair cells, and is maintained in sensory neurons and most hair cells into adulthood. Depletion of hair cell miRNAs resulting from Dicer1 conditional knockout (CKO) in Atoh1-Cre transgenic mice leads to more disparate basal-apical gene expression profiles and eventual hair cell loss. Results suggest that hair cell miRNAs subdue cochlear gradient gene expression and are required for hair cell maintenance and survival.

From zebrafish to mammal: functional evolution of prestin, the motor protein of cochlear outer hair cells.

Prestin is the motor protein of cochlear outer hair cells. It belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Members of this family serve two fundamentally distinct functions. Although most members transport different anion substrates across a variety of epithelia, prestin (SLC26A5) is unique, functioning as a voltage-dependent motor protein. Recent evidence suggests that prestin orthologs from zebrafish and chicken are electrogenic divalent/chloride anion exchangers/transporters with no motor function. These studies appear to suggest that prestin was evolved from an anion transporter. We examined the motor and transport functions of prestin and its orthologs from four different species in the vertebrate lineage, to gain insights of how these two physiological functions became distinct. Somatic motility, voltage-dependent nonlinear capacitance (NLC), and transporter function were measured in transfected human embryonic kidney (HEK) cells using voltage-clamp and anion uptake techniques. Zebrafish and chicken prestins both exhibited weak NLC, with peaks significantly shifted in the depolarization (right) direction. This was contrasted by robust NLC with peaks left shifted in the platypus and gerbil. The platypus and gerbil prestins retained little transporter function compared with robust anion transport capacities in the zebrafish and chicken orthologs. Somatic motility was detected only in the platypus and gerbil prestins. There appears to be an inverse relationship between NLC and anion transport functions, whereas motor function appears to have emerged only in mammalian prestin. Our results suggest that motor function is an innovation of therian prestin and is concurrent with diminished transporter capabilities.

The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis.

In mouse ear development, two bHLH genes, Atoh1 and Neurog1, are essential for hair cell and sensory neuron differentiation. Evolution converted the original simple atonal-dependent neurosensory cell formation program of diploblasts into the derived developmental program of vertebrates that generates two neurosensory cell types, the sensory neuron and the sensory hair cell. This transformation was achieved through gene multiplication in ancestral triploblasts resulting in the expansion of the atonal bHLH gene family. Novel genes of the Neurogenin and NeuroD families are upregulated prior to the expression of Atoh1. Recent data suggest that NeuroD and Neurogenin were lost or their function in neuronal specification reduced in flies, thus changing our perception of the evolution of these genes. This sequence of expression changes was accompanied by modification of the E-box binding sites of these genes to regulate different downstream genes and to form inhibitory loops among each other, thus fine-tuning expression transitions.

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.