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 Ca2+. In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a KD = 2.39 × 10-7m. Ca2+ at 26.5-68.0 μm promoted binding, with KD = 5.26 × 10-8m (at 61 μm Ca2+). 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 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 exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 μm Ca2+. Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells. 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 Ca2+. In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a KD = 2.39 × 10-7m. Ca2+ at 26.5-68.0 μm promoted binding, with KD = 5.26 × 10-8m (at 61 μm Ca2+). 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 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 exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 μm Ca2+. Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells. HCN1 2The abbreviations used are: HCN1, hyperpolarization-activated, cyclic nucleotide-gated nonselective cation channel isoform 1; HCN1-N, HCN1 amino-terminal cytoplasmic region; protocadherin 15 CD3-C, carboxyl-terminal sequence of protocadherin 15 CD3; 3-AT, 3-amino-1,2,4-triazole; aa, amino acid(s); PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; SPR, surface plasmon resonance.2The abbreviations used are: HCN1, hyperpolarization-activated, cyclic nucleotide-gated nonselective cation channel isoform 1; HCN1-N, HCN1 amino-terminal cytoplasmic region; protocadherin 15 CD3-C, carboxyl-terminal sequence of protocadherin 15 CD3; 3-AT, 3-amino-1,2,4-triazole; aa, amino acid(s); PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; SPR, surface plasmon resonance. is the primary full-length HCN isoform underlying Ih (hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation channel current) in a model hair cell preparation from the trout sacccule (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). cAMP-gated Ih, possibly in addition to the mechanosensory-transduction current, sets the membrane potential for a subpopulation of saccular hair cells (2Holt J.R. Eatock R.A. J. Neurophysiol... 1995; 73: 1484-1502Google Scholar, 3Farris H.E. Wells G.B. Ricci A.J. J. Neurosci... 2006; 26: 12526-12536Google Scholar). 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 (2Holt J.R. Eatock R.A. J. Neurophysiol... 1995; 73: 1484-1502Google Scholar). Given that saccular hair cells expressing IK1 in addition to Ih 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 (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). 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. 4Ramakrishnan N.A. Drescher M.J. Drescher D.G. Assoc. Res. Otolaryngol. Abstr... 2007; 30: 31Google Scholar). In general, little is known about protein-protein interactions for the HCN isoforms that would modulate Ih and/or the associated instantaneous current (5Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem... 2002; 277: 5101-5109Google Scholar). 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 (6Seiler C. Finger-Baier K.C. Rinner O. Makhankov Y.V. Schwarz H. Neuhauss S.C. Nicolson T. Development.. 2005; 132: 615-623Google Scholar), 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 (7Ahmed Z.M. Goodyear R. Riazuddin S. Lagziel A. Legan P.K. Behra M. Burgess S.M. Lilley K.S. Wilcox E.R. Riazuddin S. Griffith A.J. Frolenkov G.I. Belyantseva I.A. Richardson G.P. Friedman T.B. J. Neurosci... 2006; 26: 7022-7034Google Scholar, 8Kazmierczak P. Sakaguchi H. Tokita J. Wilson-Kubalek E.M. Milligan R.A. Müller U. Kachar B. Nature.. 2007; 449: 87-92Google Scholar). Acquisition of a Model Hair Cell Preparation from the Trout Saccule and an Organ of Corti Subfraction from the Rat Cochlea—Hair cell layers from the trout saccule as previously D.G. Drescher M.J. Hatfield J.S. Scholar, M.J. Drescher D.G. J. Scholar), a hair cell preparation of × saccular hair cells, of supporting cells. organ of Corti was from rat as previously and characterized Drescher M.J. Hatfield J.S. Drescher D.G. Res. Scholar). Trout Hair Cell two-hybrid for the proteins of trout hair cell HCN1 was the cytoplasmic amino terminus of trout saccular hair cell HCN1 as was on trout hair cell cDNA with and sites The was with and and a to a with the The to be in a necessary for of trout hair cell cDNA was from the hair cell (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar, 4Ramakrishnan N.A. Drescher M.J. Drescher D.G. Assoc. Res. Otolaryngol. Abstr... 2007; 30: 31Google for to Hair cell message was with and with an of cDNA used in the of on and the in with the cells in to for The cells to in at and on a the used to by the with and the Rat Organ of between HCN1 and protocadherin 15 CD3 was with yeast two-hybrid The HCN1 amino-terminal cytoplasmic region was from a rat organ of Corti cDNA and the sequence was and the the carboxyl terminus of protocadherin 15 CD3 in rat organ of Corti was in lower and in the the yeast two-hybrid of was to yeast the yeast two-hybrid and the on for The and on the or the with for Hair Cell model saccular hair cell preparation × hair was in CHAPS, and or and at × for at The was with with at and the was at × for and a in an the proteins to a The membrane was with for and at with the primary in to the The primary by protein the found in the carboxyl terminus of full-length HCN1 sequence (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). The and for in the with and the proteins detected from the of trout and in in on for of the saccular the and and a with and D.G. Drescher M.J. Hatfield J.S. Scholar). in the saccular in at in a and in The of the rat for analysis has been previously M.J. Drescher D.G. Hatfield J.S. N.A. Neuroscience.. 2006; Scholar). In the from from to with the of in at Drescher M.J. Hatfield J.S. Drescher D.G. Neuroscience.. 2002; Scholar). for the in for at The cochlear in a and in of trout saccule and rat in for for (for to the in the was for at The primary was for at The was used with as was with to The primary for trout saccular hair cell HCN1 was a for Hair Cell used at Rat cochlear HCN1 was immunolocalized with at the to amino acids of rat HCN1 (GenBank™ accession number The amino terminus of rat HCN1 expressed as a protein was with on was with a and with an and the was with an with and and with a and the In of localization of HCN1 in the trout saccule and rat two of the primary with or for Rat Organ of Corti and for Rat cDNA of and Hair of organ of Corti was to cDNA with or Drescher M.J. Hatfield J.S. Drescher D.G. Res. Scholar). for rat HCN1 (GenBank™ accession number and predicted to a The of HCN1 message in cochlear hair cells was from of HCN1 in to cDNA libraries cDNA with a B. Scholar, M.J. Hatfield J.S. Drescher A.J. Drescher D.G. Scholar). with an HCN1 with was by a with HCN1 for the of was to the inner hair cell with and predicted to a of the for HCN1 and Protocadherin 15 CD3 of the Rat Organ of was from a rat organ of Corti cDNA rat and to and sites and The to with a sequence of rat protocadherin 15 CD3 was with and to and sites and The with for and and and proteins expressed in of The protein for was with a or with by The protein was in a with or in at and was by a in a rat was with protein in for at In a of Ca2+ was at μm in for of and the by and in The proteins with protein at The proteins in the from at × by and to a The membrane was in for at with for in for and for in in of the membrane in the was detected with an the and with the of the and a or of Binding between HCN1 and Protocadherin 15 CD3 in Rat Organ of as D.G. N.A. Drescher M.J. with a is an for interactions in is on a Binding of the to the the of the be the of the is not In the proteins from rat organ of Corti cDNA for for the carboxyl terminus of protocadherin 15 CD3 and its deletion was used for the carboxyl terminus of protocadherin 15 (GenBank™ accession number and for rat the as for the with sites for and and for and with sites for and and proteins as in the was on a by The involved activation of on a by with by of the to the surface in the with Binding analysis was by the the cells and and the interaction between and was from the of to The of Ca2+ dependence of binding was the analysis of Ca2+ in by Protocadherin 15a-like to Trout Hair Cell HCN1, by two-hybrid protocols used to binding for HCN1, the primary HCN isoform underlying Ih in hair cells of the trout saccule (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). Amino terminus sequence in was in protocols with a saccular hair cell cDNA in for the was × of the number of that on the The was in a the in for the amino-terminal HCN1 protocadherin 15a-like protein was as a for HCN1 amino terminus and and Binding was confirmed by and at by of and amino of the trout saccular hair cell protocadherin 15a-like protein with of D. rerio (GenBank™ accession number of of the carboxyl terminus of protocadherin 15a with of amino of the carboxyl terminus of protocadherin 15a for trout and D. of two binding and sequence in the trout saccular hair cell The sequence for the trout saccular hair cell protocadherin 15a-like protein to the cytoplasmic carboxyl terminus of protocadherin 15a of D. with and the homology to D. rerio protein. The trout sequence was in with a in to the for D. rerio protocadherin 15a. The trout protein two and sequence and The homology in amino sequence between the trout hair cell and D. rerio protocadherin 15a was and for of The homology of the trout saccular hair cell sequence to the carboxyl terminus sequence of D. rerio protocadherin was and Binding between and the protocadherin 15a-like protein was by the of the sequence in the protein that was not involved in the The carboxyl terminus of protocadherin 15a is the cytoplasmic of tip link protein, would be for protein-protein binding, for the protocadherin 15a-like protein and HCN1 expressed in hair cells of the trout of HCN1 in Hair of the Trout was found in the carboxyl terminus of HCN1 expressed in the model hair cell preparation from the trout for the hair cell is the cell (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). As the by protein a protein in the trout saccular hair cell preparation on with the of the predicted for HCN1 (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google (GenBank™ accession number the primary saccular hair cells in the detected with or was at sites of hair cells, to the and cell membrane are sites of D.G. N.A. Drescher M.J. Hatfield J.S. Neuroscience.. Scholar). In HCN1 was immunolocalized to stereocilia, with discrete sites of that gradated in distribution and HCN1 in and Hair message was detected in the cochlear cDNA and in cDNA libraries of inner and outer hair cells The outer hair cell and inner hair cell the characterized by HCN1 HCN1 protein was immunolocalized with a the amino terminus of rat HCN1 and a protein of organ of Corti on HCN1 was to and the to the organ of the organ of Corti to the of inner hair cells and outer hair cells and and in consistent with HCN1 in cell for HCN1 protein was also found to be associated with hair cell stereocilia, both of inner hair cells and outer hair cells with by of the primary with the organ of Corti of the rat HCN1 is immunolocalized to and the to the of the inner hair cell was also to stereocilia of the inner hair cell and outer hair cell HCN1 is associated with stereocilia of outer hair cells as as inner hair cells and the and to sites to the of the inner hair cell is at the of outer hair cells cells and is associated with the inner hair cell stereociliary array sites of the inner hair cell from E.A. Scholar). of HCN1 associated with the stereociliary array of an inner hair of the Amino of Rat Organ of Corti HCN1 with the of Organ of Corti Protocadherin 15 CD3 in of HCN1 in rat cochlear hair cells and of HCN1 protein to stereocilia of inner and outer hair cells, to of HCN1 protein to stereocilia of hair cells in the trout we binding of HCN1 to protocadherin 15 CD3. Protocadherin 15 CD3 is to be a of protocadherin 15a of D. the implicated as a binding for HCN1 in trout saccular hair cells. The amino-terminal for rat organ of Corti HCN1 comparable with the amino-terminal of trout saccular hair cell HCN1 was found by yeast two-hybrid protocols to bind the carboxyl terminus of protocadherin 15 CD3 expressed in the rat organ of Corti The of used for analysis the trout protocadherin 15a-like carboxyl terminus amino sequence as binding in yeast two-hybrid protocols and to of rat protocadherin 15 CD3 (GenBank™ accession number Binding with rat organ of Corti as and as or the with as and as at was found in in of Binding between HCN1 and Protocadherin 15 CD3 from Rat Organ of Corti with yeast two-hybrid analysis was confirmed with a pull-down a predicted for rat organ of Corti protein of detected with for rat HCN1 or with an the on the protein Binding between rat organ of Corti and rat organ of Corti was of and of The binding was with of Ca2+ at with or with the as for the of HCN1 Binding to Protocadherin 15 CD3 for Organ of Corti amino terminus of rat organ of Corti HCN1 and the carboxyl terminus of rat organ of Corti protocadherin 15 CD3 and found to bind in the presence of as and as the KD = × 10-7m. binding was in the presence of a KD = × 10-7m. The binding of to was by by at 26.5 μm Ca2+ to binding with with in binding to 68 μm Ca2+ and Binding at 68 μm Ca2+ was than binding at 26.5 μm Ca2+ analysis, binding was detected for and Binding as a of in the presence of 61 μm Ca2+ a KD = × and in a of at 61 μm a KD of 5.26 × 10-8m consistent with of binding between and with and as and as binding was between and CD3-C, was promoted by Ca2+ The KD for interaction was × 10-8m from a as 61 μm Ca2+). mutants for and the proteins 26.5 μm binding with was with deletion with deletion with binding for deletion binding than for the and The in binding between and with HCN1-N, to a region in protocadherin 15 CD3, accession number as for binding to region in rat protocadherin 15 CD3, implicated as for binding to rat HCN1, is in amino sequence to the trout protocadherin 15a-like protein than that found for rat with the trout protocadherin 15a-like protein or D. rerio protocadherin 15a. of Binding to the of Protocadherin 15 from Rat Organ of 15 is a protocadherin 15 protein implicated as a role in the of hair cell tip links (7Ahmed Z.M. Goodyear R. Riazuddin S. Lagziel A. Legan P.K. Behra M. Burgess S.M. Lilley K.S. Wilcox E.R. Riazuddin S. Griffith A.J. Frolenkov G.I. Belyantseva I.A. Richardson G.P. Friedman T.B. J. Neurosci... 2006; 26: 7022-7034Google Scholar, 8Kazmierczak P. Sakaguchi H. Tokita J. Wilson-Kubalek E.M. Milligan R.A. Müller U. Kachar B. Nature.. 2007; 449: 87-92Google Scholar). The carboxyl terminus of rat protocadherin 15 with homology to rat protocadherin 15 CD3 or binding to the amino terminus of HCN1 at 26.5 μm Ca2+ or 61 μm Ca2+ of Binding to from Rat Organ of SPR, evidence was for binding of as to as maximum response was at 26.5 μm Ca2+ with that at 61 μm Ca2+ and response was in the of Ca2+ The response at μm Ca2+ was lower than that at 26.5 μm Ca2+ analysis, for and lower than for and in at 61 μm Ca2+ and The KD for the interaction between and at 61 μm Ca2+ = × HCN of the channel underlie Ih, a cation Ih has for to is by the cation in The for Ih from expressed HCN1 are reported to be and for and to of and A. M. M. Nature.. Scholar). Ih in or in and also Ca2+ with Ca2+ current of current U. S. Scholar, P. T. B. H. J. 2007; Scholar). In addition to HCN also for a instantaneous current, HCN isoforms are expressed in cells (5Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem... 2002; 277: 5101-5109Google Scholar, R. R. Nature.. Scholar, M. A. H. J. Biol. Chem... Scholar, R. A. R. A. P. U. S. Scholar, R. J. 2003; Scholar, V. Accili E.A. J. Biol. Chem... Scholar, E.M. T. E. R.A. 2005; Scholar, P. A. M. J. 2006; or in J. J.S. M. M. U. S. Scholar). with the of the to Ih in the with both in V. Accili E.A. J. Biol. Chem... Scholar). the and Ih to of the channel V. Accili E.A. J. Biol. Chem... or of C. J. 2006; is In Ih has been detected in hair cells of vestibular of both lower and (2Holt J.R. Eatock R.A. J. Neurophysiol... 1995; 73: 1484-1502Google Scholar, T. J. Scholar, S. M.J. J. Neurophysiol... Scholar, A. A. Eatock R.A. J. Neurosci... as as in cells from the organ of Corti of the J. Scholar). In and a subpopulation of hair cells Ih as cation is hair cells the hair cells both IK1 and that membrane sufficiently depolarized to activation of voltage-gated calcium channels, influx of and of hair cell transmitter (2Holt J.R. Eatock R.A. J. Neurophysiol... 1995; 73: 1484-1502Google Scholar). previously of for HCN isoforms in a model hair cell preparation from the trout evidence that HCN1 is the primary full-length HCN isoform expressed in saccular hair cells (1Cho W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). for (GenBank™ accession and and are Ih in hair cells of the saccule would therefore from expressed HCN1, with HCN1 channel for hair spontaneous activity in the saccule in Ref. W.J. Drescher M.J. Hatfield J.S. Bessert D.A. Skoff R.P. Drescher D.G. Neuroscience.. 2003; 118: 525-534Google Scholar). role for Ih would at of and B. H. D. E.R. Scholar), and in for trout Scholar). However, hair cells, the cells in both vestibular and auditory are In addition to at sites by hair cells, stereocilia cell cytoplasmic an in and in found in the inner of lower and at for the saccule of O. Scholar), and for the saccule of Biol. Scholar). in the rat that at at and at cochlear O. E. C. J. Scholar). in would be predicted to the potential for Ih to V. Accili E.A. J. Biol. Chem... for HCN1 to the hair cell stereocilia. In the yeast two-hybrid protocols to in a model hair cell preparation from the trout saccule D.G. Drescher M.J. Hatfield J.S. Scholar), binding for hair cell HCN1 N.A. Drescher M.J. Drescher D.G. Assoc. Res. Otolaryngol. Abstr... 2007; 30: 31Google Scholar). of for binding to the amino terminus of HCN1 as a protocadherin 15a-like protein, implicated as a tip link protein in D. rerio (6Seiler C. Finger-Baier K.C. Rinner O. Makhankov Y.V. Schwarz H. Neuhauss S.C. Nicolson T. Development.. 2005; 132: 615-623Google Scholar). of and and with confirmed interaction of the amino cytoplasmic terminus of HCN1 with the carboxyl cytoplasmic terminus of the protocadherin 15a-like protein. The of the hair cell protein as a protocadherin 15a-like protein was the of between the trout and D. rerio splice variants for the carboxyl terminus of protocadherin 15 are known to exist in D. with protocadherin 15a and to than D. rerio protocadherin 15a to the trout The trout sequence was in with a as the D. rerio protocadherin 15a The that was the carboxyl terminus of protocadherin 15a that was the cytoplasmic of protocadherin 15a for protein-protein was also of interaction was by for of and and for protocadherin 15a-like in the of HCN1. The of HCN1 to hair cell stereocilia with trout saccular hair cell HCN1, at sites to of predicted tip link was a of supporting interaction between an HCN1 channel and a tip link protein in the saccular hair the that for HCN1 was expressed in cochlear hair cells and HCN1 protein be immunolocalized to cochlear hair cell stereocilia, that organ of Corti HCN1 would bind to protocadherin 15 as found for the model hair cell preparation from the trout protein-protein interaction between the amino terminus of HCN1 and carboxyl cytoplasmic terminus of protocadherin 15 CD3 was confirmed for the rat organ of Corti with yeast two-hybrid and with of binding and interaction was for the binding and binding in the model pointed to a dependence of the 15 CD3 interaction on calcium with the maximum and binding in the presence of Ca2+. amino binding sites for Ca2+ be in the rat protocadherin 15 CD3 carboxyl terminus possibly to the calcium of binding by mutants of the carboxyl terminus of rat organ of Corti protocadherin 15 CD3 to a region as for binding, a region that homology to the comparable region of protocadherin 15a-like protein expressed in trout saccular hair cells. In to the interaction between HCN1 and protocadherin 15 CD3, little to binding between the amino terminus of rat organ of Corti HCN1 and the carboxyl terminus of protocadherin 15 the region of protocadherin 15 CD3. analysis that rat organ of Corti HCN1 also to its amino and a for channel from HCN1 The amino terminus binding by was with the of in the amino terminus The interaction between and from the interaction between and protocadherin 15 CD3 in that binding was between HCN1 in the of Ca2+. binding was to lower of Ca2+ for HCN1 binding to to HCN1 binding to protocadherin 15 CD3. The would be consistent with a between binding and binding, from 26.5 to 68 μm Ca2+. Given interaction between protocadherin 15 CD3, a hair cell tip link protein to be involved in mechanosensory and HCN1, the as to the possible of the channel in Ih is and would not be a for the mechanosensory transduction channel on the of response of mechanosensory transduction in hair cells A.J. 26: Scholar), although activation of Ih by has been with both activation and U. 2007; Scholar). for by of the HCN isoforms is both and and is with Ih by C. J. 2006; Scholar, K.S. J. Scholar). of the HCN instantaneous current is that is by P. A. M. J. 2006; Scholar), with possible to spontaneous of hair associated with mechanosensory transduction P. D. A.J. J. Neurosci... 2003; Scholar). the instantaneous current is detected at V. Accili E.A. J. Biol. Chem... Scholar), as would be in structural that the hair cell mechanosensory transduction be on the of the shorter A.J. Kachar B. J. S.M. J. 2006; Scholar, B. M. M. U. S. Scholar), and is the of shorter stereocilia that to the of protocadherin 15 as to the of tip links with stereociliary insertion the taller stereocilia P. Sakaguchi H. Tokita J. Wilson-Kubalek E.M. Milligan R.A. Müller U. Kachar B. Nature.. 2007; 449: 87-92Google Scholar). to the tips of the shorter stereocilia as sites for calcium in the of mechanosensory consistent with of mechanosensory transduction and protocadherin 15 tip link insertion sites C. Assoc. Res. Otolaryngol. Abstr... Scholar, A. C. Assoc. Res. Otolaryngol. Abstr... Scholar, R. Assoc. Res. Otolaryngol. Abstr... Scholar). evidence in the to HCN1 as binding the tip link protocadherin 15 CD3, a role for HCN1 in hair cell mechanosensory transduction. for the of the and of for of Ca2+ and with