Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca2+-dependent manner (with optimal 61 μm free Ca2+ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca2+, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (Kd = 1.7 × 10–5m) than for the C2F interaction (Kd = 2.6 × 10–9m). In contrast, it was the otoferlin C2D domain that bound the Cav1.3 II-III loop, in a Ca2+-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca2+ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function. The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca2+-dependent manner (with optimal 61 μm free Ca2+ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca2+, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (Kd = 1.7 × 10–5m) than for the C2F interaction (Kd = 2.6 × 10–9m). In contrast, it was the otoferlin C2D domain that bound the Cav1.3 II-III loop, in a Ca2+-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca2+ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function. Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref. 1Südhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1837) Google Scholar). In mechanosensory hair cells, calcium microdomains (2Tucker T. Fettiplace R. Neuron. 1995; 15: 1323-1335Abstract Full Text PDF PubMed Scopus (132) Google Scholar) and possibly nanodomains (3Brandt A. Khimich D. Moser T. J. Neurosci. 2005; 25: 11577-11585Crossref PubMed Scopus (224) Google Scholar) are formed when voltage-gated calcium channels open upon depolarization. Calcium at these sites is thought to activate protein interactions, leading to vesicle fusion. Some of the key players in this process are the target-SNARE 2The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; 3-AT, 3-amino-1,2,4-triazole; GST, glutathione S-transferase; HBS-E, HEPES-buffered saline with EDTA; HBS-EP, HEPES-buffered saline with EDTA and phosphate; HBS-P, HEPES-buffered saline with phosphate; IPTG, isopropyl β-d-1-thiogalactopyranoside; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with Tween 20; RU, response unit; SPR, surface plasmon resonance.2The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; 3-AT, 3-amino-1,2,4-triazole; GST, glutathione S-transferase; HBS-E, HEPES-buffered saline with EDTA; HBS-EP, HEPES-buffered saline with EDTA and phosphate; HBS-P, HEPES-buffered saline with phosphate; IPTG, isopropyl β-d-1-thiogalactopyranoside; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with Tween 20; RU, response unit; SPR, surface plasmon resonance. proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin (4Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Vesicle-SNARE synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal synapse, modulating calcium channels and vesicle release by a Ca2+-dependent interaction with other SNARE proteins in the presence of lipid molecules (4Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 5Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar, 6Tang J. Maximov A. Shin O. Dai H. Rizo J. Südhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not detected (7Safieddine S. Wenthold R.J. Euro. J. Neurosci. 1999; 11: 803-812Crossref PubMed Scopus (128) Google Scholar). Instead, fast neurotransmitter release in auditory and vestibular hair cells, facilitated largely by an L-type voltagegated calcium channel, Cav1.3 (8Lewis R.S. Hudspeth A.J. Nature. 1983; 304: 538-541Crossref PubMed Scopus (255) Google Scholar, 9Platzer J. Engel J. Schrott-Fischer A. Stephan K. Bova S. Chen H. Zheng H. Striessing J. Cell. 2000; 102: 89-97Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar), is thought to be modulated by a newly discovered protein, otoferlin, acting as the Ca2+ sensor and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic deafness (10Yasunaga S. Grati M. Cohen-Salmon M. El-Amraoui A. Mustapha M. Salem N. El-Zir E. Loiselet J. Petit C. Nat. Genet. 1999; 21: 363-369Crossref PubMed Scopus (402) Google Scholar). Gene sequences of different deaf families show that the OTOF gene can undergo mutation at multiple locations (11Migliosi V. Modamio-Høybjør S. Moreno-Pelayo M.A. Rodríguez-Ballesteros M. Villamar M. Tellería D. Menéndez I. Moreno F. del Castillo I. J. Med. Genet. 2002; 39: 502-506Crossref PubMed Google Scholar, 12Tekin M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar, 13Yasunaga S. Grati M. Chardenoux S. Smith T.N. Friedman T.B. Lalwani A.K. Wilcox E.R. Petit C. Am. J. Hum. Genet. 2000; 67: 591-600Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Recently, it has been demonstrated that otoferlin is necessary for synaptic exocytosis from hair cells (14Roux I. Safieddine S. Nouvian R. Grati M. Simmler M. Bahloul A. Perfettini I. Rostaing P. Hamard G. Triller A. Avan P. Moser T. Petit C. Cell. 2006; 127: 277-289Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Further, an engineered mutation in the C2B domain of otoferlin has been shown to cause deafness in mice (15Longo-Guess C. Gagnon L.H. Bergstrom D.E. Johnson K.R. Hear. Res. 2007; 234: 21-28Crossref PubMed Scopus (42) Google Scholar). However, the precise function of otoferlin as a synaptic protein is not well understood. Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in humans have been documented to cause DFNB9 deafness (11Migliosi V. Modamio-Høybjør S. Moreno-Pelayo M.A. Rodríguez-Ballesteros M. Villamar M. Tellería D. Menéndez I. Moreno F. del Castillo I. J. Med. Genet. 2002; 39: 502-506Crossref PubMed Google Scholar, 12Tekin M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar). Previous studies suggested that a region of otoferlin containing all three C2 and to the molecules syntaxin 1A and SNAP-25 in response to an in Ca2+ (14Roux I. Safieddine S. Nouvian R. Grati M. Simmler M. Bahloul A. Perfettini I. Rostaing P. Hamard G. Triller A. Avan P. Moser T. Petit C. Cell. 2006; 127: 277-289Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). However, it is not a amino acid in domain of otoferlin, as C2F (11Migliosi V. Modamio-Høybjør S. Moreno-Pelayo M.A. Rodríguez-Ballesteros M. Villamar M. Tellería D. Menéndez I. Moreno F. del Castillo I. J. Med. Genet. 2002; 39: 502-506Crossref PubMed Google Scholar) or C2D M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar), to we the role of otoferlin as a Ca2+ sensor as well as a of vesicle as by interactions and for fusion proteins, and and the were from fusion and HBS-E, HBS-P, and for surface plasmon were from and and were from phosphate-buffered saline and were from of C2 1A, SNAP-25, and Cav1.3 II-III in for otoferlin C2 syntaxin 1A the SNAP-25, and the Cav1.3 II-III loop, containing the were from and and used in containing as were with and The were in cells were used for otoferlin otoferlin otoferlin otoferlin otoferlin otoferlin syntaxin 1A, SNAP-25, and the Cav1.3 II-III loop, C2 the otoferlin C2 domains were generated in in two by The and mutated otoferlin for and otoferlin for In the of the mutated was with the or C2D or C2F and otoferlin to two that the mutated, at In the the were in and to for three of C2D or C2F mutated C2D or C2F and were and the was The were and for of the amino acid change, in of containing the sequences were used to E. were and of fusion proteins was induced by 1 isopropyl in The were for at The cells were in and with protein by that were and for of fusion proteins, of were for with protein induced by The cells were and and the a acid affinity The were with three to and the proteins were with of the were in an of and containing with or for at and the of the the proteins were from the and protein was with the of the proteins was by with and In an not cells induced with were by by The was with of The were and and the bound fusion proteins were by the of by and The containing the fusion proteins was containing of 1A and Cav1.3 II-III in for of fusion proteins were for for syntaxin 1A and Cav1.3 II-III containing the sites were used in to syntaxin and the calcium domain in of containing syntaxin 1A or the Cav1.3 II-III were to E. The were for of the fusion protein by cell were used in or the fusion were and were used to and were in a to the The that were a containing and for at or were with for binding were fusion proteins 1A and II-III and fusion proteins C2 were with C2 domains in binding for at of a were to the for 1 at by for at × The were in the binding at for in and The was in a to a and was with a fusion proteins were a by an S. G. PubMed Scopus Google Scholar). Biol. PubMed Scopus Google Scholar) were protein with different of otoferlin C2 domain fusion protein The surface was with and The response was as the the binding and were binding studies were of the and binding of the interaction of otoferlin C2 domains with syntaxin 1A, SNAP-25, and the Cav1.3 II-III loop, the fusion proteins were as a C2D and C2F domains were to a of in with 61 μm Ca2+ for optimal binding. were and the were with the Biol. PubMed Scopus Google Scholar). binding was However, when was a P. J. Full Text PDF PubMed Scopus Google Scholar) in the binding with was The M. P. I. 1993; PubMed Scopus Google Scholar) were and the with the were the of the binding from that of the and were in the of we the F. J. 1999; Scholar), in all the the different were with the of and were also to the of C2F and in were in a with at and the was from to were with a at a The were in to a of and the was from the of Ca2+ of all were by because of the of Ca2+ in the Ca2+ in the present are C2 of of otoferlin, from amino acid substitutions and by M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar), are in DFNB9 patients otoferlin in humans and the is not the or the of the otoferlin is in the are that the are S. Grati M. Chardenoux S. Smith T.N. Friedman T.B. Lalwani A.K. Wilcox E.R. Petit C. Am. J. Hum. Genet. 2000; 67: 591-600Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). C2F to studies otoferlin domains and as in were in syntaxin 1A fusion protein, was as in In containing 3-AT, that syntaxin 1A interaction with and C2F. were present when C2D and were used as When and C2F were in a containing 3-AT, for C2F and were or was with with 1A in 1A fusion protein and domain fusion proteins that syntaxin 1A and as well as C2F However, it is the C2F interaction that is Ca2+-dependent the interaction for and Ca2+ is not of 1A of the otoferlin C2 domains with syntaxin 1A was also with SPR, fusion proteins of the C2 domains as and syntaxin 1A fusion protein a sensor as The C2 domains were to The C2F domain bound syntaxin 1A in was binding when was used as a Ca2+ of C2F with the binding of the C2F fusion protein to syntaxin 1A at Ca2+ binding was when C2F was in containing However, a was when the C2F binding was in containing μm free Ca2+ in free Ca2+, was a in binding that at 61 μm Ca2+ However, was a of than in the binding when the Ca2+ was to μm and was in binding when the of Ca2+ was to and μm and The were by the response for and from a in of the were multiple and for different Ca2+ were was binding to the cell was by the present that the C2F domain of otoferlin interacts with syntaxin this interaction free Ca2+ in the of and is interaction Ca2+ is The C2F domain is the shown in 1 and be to in a manner in other of C2 domains in a binding was to Ca2+, the domain was to in containing μm free Ca2+ or EDTA and by was the that was in binding when free Ca2+ was (with present it that the interaction is insensitive to Ca2+, in to the calcium of the C2F C2B and not Ca2+ for binding to syntaxin 1A in binding and RU, P1825A in the C2F C2F Ca2+ a amino acid in the C2F domain with nonsyndromic deafness in humans (11Migliosi V. Modamio-Høybjør S. Moreno-Pelayo M.A. Rodríguez-Ballesteros M. Villamar M. Tellería D. Menéndez I. Moreno F. del Castillo I. J. Med. Genet. 2002; 39: 502-506Crossref PubMed Google Scholar), we the same amino acid (P1825A) in that is to cause deafness in humans, by in C2F. The fusion protein interaction with syntaxin 1A in and in to the binding of native C2F to syntaxin 1A and C2F native and mutated also demonstrated that the not with syntaxin 1A The of the P1825A mutation in C2F and of the with a in the for native C2F. Calcium of the C2F of the C2F domain by and other amino was with Ca2+ for native but not the mutated and of show in the presence of 61 μm free Ca2+ and in the of Ca2+ by the that the not in in the presence of 61 μm free Ca2+ compared with the of free However, for the native C2F is by the of Ca2+ μm free Ca2+ was by the native of C2 fusion protein was with by we with the C2F domain fusion protein The that fusion proteins as C2F with were in the present The show a at as well as a for a in the The was and Ca2+ of C2F to binding SNAP-25 as and native and mutated C2F domains as that the native C2F SNAP-25 the mutated, not that the binding is to is binding when is free Ca2+ However, when the free Ca2+ to is an in binding and a in binding when the free Ca2+ to μm An in binding the free Ca2+ 61 μm when the free Ca2+ is the binding by and to to a free Ca2+ of μm C2D to the Calcium Cav1.3 II-III otoferlin C2 domains were in and with the Cav1.3 II-III were used to In containing 3-AT, the C2D with Cav1.3 II-III the C2D with the not In the of that the the of the we also detected interaction of Cav1.3 with the and C2F the interactions were weaker II-III interaction was also demonstrated in native C2D bound the II-III loop, a In contrast, the mutated not bind the II-III The fusion proteins of the otoferlin C2 domains were for interaction with the Cav1.3 II-III C2 domains in binding were as C2D the response other C2 domains interaction C2F C2B and RU, not was response when 1 was used as a a fusion protein with an amino acid in to that in DFNB9 deafness M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar), we binding to the Cav1.3 II-III SPR, we detected that the binding was compared with the native II-III we detected that the otoferlin interaction in the presence of free An in free Ca2+ this Ca2+ (with the Cav1.3 II-III a sensor and C2D in containing a of free Ca2+ was binding with free Ca2+ However, a in the binding when the free Ca2+ was to 61 μm that Ca2+ the interaction in in binding at the Ca2+ In contrast, we that for binding to the Cav1.3 II-III loop, free Ca2+ is was in binding with an in free Ca2+ the Cav1.3 interacts with otoferlin the C2D domain with native C2D an of 61 μm free syntaxin 1A as we the binding of otoferlin domains C2F not at and with the of for of C2F containing 61 μm was Biol. PubMed Scopus Google Scholar) in the as by C2F at and the for the of C2F were to a binding to the and we a of for we than of the to the The for syntaxin syntaxin binding from suggested weaker interaction for the binding with 1 in the binding and the same of C2F as in binding of C2F to syntaxin C2 interaction 1A × × × 1A × × × × × × × × × in a SNAP-25 as in we also the binding for and of otoferlin C2F containing 61 μm Ca2+ was were as for we a of for we than The for of the interaction was to be 1.7 × II-III of the L-type voltage-gated calcium Cav1.3 was used as the in to the binding for the otoferlin C2D domain were as of × for the for domain interaction was as a Ca2+ domains in of proteins, as by synaptotagmin 1 and protein (reviewed in Ref. PubMed Scopus Google Scholar). Otoferlin, in as well as in mechanosensory hair cells N. C. Engel J. H. P. N. M. H. M. J. Neurosci. 2006; PubMed Scopus Google Scholar), C2 domains and is to be the main Ca2+ sensor of the hair cell for the OTOF gene are deaf and show exocytosis from the inner hair cells (14Roux I. Safieddine S. Nouvian R. Grati M. Simmler M. Bahloul A. Perfettini I. Rostaing P. Hamard G. Triller A. Avan P. Moser T. Petit C. Cell. 2006; 127: 277-289Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). In a of OTOF gene mutations cause of the protein (10Yasunaga S. Grati M. Cohen-Salmon M. El-Amraoui A. Mustapha M. Salem N. El-Zir E. Loiselet J. Petit C. Nat. Genet. 1999; 21: 363-369Crossref PubMed Scopus (402) Google Scholar, 13Yasunaga S. Grati M. Chardenoux S. Smith T.N. Friedman T.B. Lalwani A.K. Wilcox E.R. Petit C. Am. J. Hum. Genet. 2000; 67: 591-600Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) or amino acid substitutions in C2F (11Migliosi V. Modamio-Høybjør S. Moreno-Pelayo M.A. Rodríguez-Ballesteros M. Villamar M. Tellería D. Menéndez I. Moreno F. del Castillo I. J. Med. Genet. 2002; 39: 502-506Crossref PubMed Google Scholar), C2D M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar), or F. M. F. Petit C. S. N. 2002; PubMed Scopus Google Scholar) In the present study, we two amino acid in C2F and in to these domains may with the proteins in fusion in the hair Ca2+ of C2 demonstrated that otoferlin interacts with the synaptic proteins syntaxin 1A and SNAP-25 and that this interaction free Ca2+, as is the for the Ca2+ sensor synaptotagmin neuronal exocytosis Biol. Cell. 2007; PubMed Scopus Google Scholar). However, otoferlin may also with syntaxin 1A in a manner for C2 binding the domain and syntaxin 1A show that Ca2+ is not necessary for the that is to to the exocytosis at the hair cell the C2F domain interaction with syntaxin 1A or SNAP-25 that optimal binding is when the free Ca2+ 61 μm but that binding when the Ca2+ is The from the of an (14Roux I. Safieddine S. Nouvian R. Grati M. Simmler M. Bahloul A. Perfettini I. Rostaing P. Hamard G. Triller A. Avan P. Moser T. Petit C. Cell. 2006; 127: 277-289Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) the of binding of syntaxin 1A to an otoferlin containing the of and C2F domains to 1 Ca2+, with a of be by the that we the otoferlin C2F the (14Roux I. Safieddine S. Nouvian R. Grati M. Simmler M. Bahloul A. Perfettini I. Rostaing P. Hamard G. Triller A. Avan P. Moser T. Petit C. Cell. 2006; 127: 277-289Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) a containing the three otoferlin exocytosis in cells synaptotagmin 1 a with exocytosis at μm Ca2+ S. Shin Südhof T.C. J. 2002; 21: PubMed Scopus Google Scholar), a response to also be that different synaptotagmin show different affinity for Ca2+, as from binding as well as exocytosis S. Shin Südhof T.C. J. 2002; 21: PubMed Scopus Google Scholar). In hair cells, Ca2+-dependent exocytosis μm Ca2+ and exocytosis μm Ca2+ D. T. 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PubMed Scopus Google Scholar). and the of for and native C2F fusion proteins in the presence and of the mutated domain is insensitive to Ca2+, compared with the native C2F and in the C2 domains are from in the C2 Calcium of is the C2 domain the and in the different of the for the mutated show that is the of calcium not the that the not to bind Ca2+ and the tertiary In to the C2F the C2D domain Ca2+-dependent of in the presence of Ca2+ The that Ca2+-dependent of the for the fusion proteins in the in to the of to the different that the proteins were to to native The of in with the presence of C2F as in The can be as a of for and N. 1999; PubMed Scopus Google Scholar). is of to that N. 2000; PubMed Scopus Google Scholar). not it is that an of molecular be by the interaction of otoferlin C2 domains with the synaptic The of induced structural S. S. A. PubMed Scopus Google Scholar, R. S. A. PubMed Scopus Google Scholar) may be to the of C2 domains with binding can be that the C2F domain is the main otoferlin that interacts with SNARE be the C2 C2 domain may have a and role in the also be that the in studies not the of the lipid has been shown to Ca2+ of exocytosis in the of synaptotagmin 1 J. Maximov A. Shin O. Dai H. Rizo J. Südhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). C2 factor for exocytosis be the C2 domain binding affinity for the SNARE C2 domain of otoferlin may have a different in of as well as C2 domain or domains bind and to vesicle and in study, we compared the C2D and C2F affinity for syntaxin 1A and that the C2F syntaxin than C2D C2D with the L-type Calcium is that synaptotagmin the of otoferlin in interacts with and voltage-gated calcium channels R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the present we that otoferlin C2D domain to the II-III of is the L-type calcium in hair cells J. Engel J. Schrott-Fischer A. Stephan K. Bova S. Chen H. Zheng H. Striessing J. Cell. 2000; 102: 89-97Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). also that this interaction is by Ca2+ and that a of 61 μm Ca2+ is required for optimal binding in a mutation (L1011P) that causes deafness in humans M. Akcayoz D. Incesulu A. Am. J. Med. Genet. 2005; 138A: 6-10Crossref Scopus (48) Google Scholar) the C2D binding to Cav1.3 and the C2D is insensitive to of the otoferlin C2 C2D the Cav1.3 II-III with the other C2 domains may also be is the of the present to all interactions of of the otoferlin Ca2+-dependent with the calcium and SNARE studies Ca2+-dependent and binding of synaptotagmin to and J. Neurosci. PubMed Google Scholar) that synaptotagmin binding to syntaxin 1A as the calcium The also in the binding when of the synaptotagmin protein are syntaxin binding in the of as well as a in binding in response to an in calcium In that otoferlin C2 domains may of other in the Otoferlin, C2 two key SNARE synaptic proteins of hair cells, syntaxin 1A and interacts with syntaxin 1A the C2F mutation in C2F Ca2+ binding ability as well as interaction with syntaxin SNAP-25, protein, also interacts with the C2F that the C2D domain of otoferlin interacts with the calcium Cav1.3 in a manner and that a mutation in C2D to a binding to of otoferlin, C2 may be an to Ca2+-dependent synaptic in hair for of the