Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor ( SNARE) Proteins Required for Hair Cell Exocytosis*
Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor ( SNARE) Proteins Required for Hair Cell Exocytosis*
复制标题
DOI：
10.1074/jbc.m113.480533
复制
发表时间：
2014-03-28
影响因子：
4.8
通讯作者：
Drescher, Dennis G.
中科院分区：
文献类型：
--
作者：
Ramakrishnan, Neeliyath A.;Drescher, Marian J.;Drescher, Dennis G.
背景：耳铁蛋白缺乏损害毛细胞胞吐作用并导致耳聋。结果如下：Otoferlin结合Ca 2+，调节磷脂酰肌醇4，5-二磷酸，t-SNARE和分子内C2相互作用; otoferlin直接与SNAP-25和syntaxin-1B相互作用。结论：低钙促进耳铁蛋白C2C、C2 D、C2 E和C2F之间的相互作用;高钙驱动t-SNARE和脂质相互作用。重要性：Otoferlin是一种含有C2结构域的ferlin家族蛋白，其突变可导致人类非综合征性听力损失（DFNB 9耳聋）。此外，耳蜗内毛细胞的递质分泌在缺乏otoferlin的小鼠中受到损害。在本研究中，我们表明，C2F结构域的otoferlin直接结合钙（KD = 267米）减少结合pachanga（D1767 G）C2F小鼠突变。发现钙差异调节otoferlin C2结构域与靶SNARE（t-SNARE）蛋白和磷脂的结合。C2 D-F结构域与突触融合蛋白-1 t-SNARE基序相互作用，在20-50 m Ca 2+范围内具有最大结合。在20 m Ca 2+时，解离速率显著降低，表明与0 m Ca 2+（K-D =约10（-8））相比，结合增加（K-D =约10（-9）），表明C2结构域-SNARE复合物的钙介导稳定。C2 A和C2B与t-SNARE的相互作用对钙不敏感。C2F结构域直接结合t-SNARE SNAP-25，最大值在100 m处，在0 m Ca 2+处减少，重复C2F结构域与磷脂酰肌醇4，5-二磷酸相互作用的模式。相反，C2F不结合囊泡SNARE蛋白小突触泡蛋白-1（VAMP-1）。此外，靶向otoferlin的抗体免疫沉淀syntaxin-1和SNAP-25，但不synaptobrevin-1。与钙增加时结合增加相反，耳铁蛋白C2F结构域和分子内C2结构域之间的相互作用在不存在钙的情况下发生，与C2结构域内相互作用一致，在低钙时形成封闭的三级结构，随着钙增加而打开。这些结果表明，在胞吐和钙依赖性膜融合的调制的otoferlin的直接作用。
Background: Otoferlin deficiency impairs hair cell exocytosis and causes deafness. Results: Otoferlin binds Ca2+, regulating phosphatidylinositol 4,5-bisphosphate, t-SNARE, and intramolecular C2 interactions; otoferlin directly interacts with SNAP-25 and syntaxin-1B. Conclusion: Low Ca2+ promotes interactions among otoferlin C2C, C2D, C2E, and C2F; high Ca2+ drives t-SNARE and lipid interactions. Significance: Otoferlin may mediate novel modes of Ca2+-driven membrane fusion in hair cells.Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K-D = 267 m) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D-F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20-50 m Ca2+. At 20 m Ca2+, the dissociation rate was substantially lower, indicating increased binding (K-D = approximate to 10(-9)) compared with 0 m Ca2+ (K-D = approximate to 10(-8)), suggesting a calcium-mediated stabilization of the C2 domaint-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 m and with reduction at 0 m Ca2+, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a closed tertiary structure at low calcium that opens as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.