Deciphering the properties and function of calcium binding proteins (CaBPs) in synaptic sound encoding

破译突触声音编码中钙结合蛋白（CaBP）的特性和功能

• 批准号: 279586126
• 负责人: Professorin Dr. Tina Pangrsic Vilfan
• 金额: --
• 依托单位: Klinik für Hals-Nasen-Ohrenheilkunde
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/279586126?language=en
• 关键词: Deciphering; properties; function; calcium; binding

Voltage-gated CaV1.3 (L-type) Ca2+ channels mediate Ca2+ influx and exocytosis at the auditory inner hair cell (IHC) ribbon synapse and are required for hearing. These channels undergo a negative feedback regulation by incoming Ca2+ ions (Ca2+-dependent inactivation) that is mediated by calmodulin. The action of calmodulin is prevented by Ca2+ binding proteins (CaBPs) that, together with calmodulin, belong to the large family of EF-hand-motif-containing proteins. Previously, an expression of CaBP1, 2, 4, and 5 in mouse cochlear IHCs has been demonstrated. Whereas genetic deletion of CaBP4 causes only a minor alteration of IHC Ca2+ influx leaving hearing unperturbed, mutations in CaBP2 have recently been identified to underlie a nonsyndromic form of autosomal recessive deafness (DFNB93) in human patients. An ongoing study currently investigates the cellular disease mechanisms of this hearing disorder in a mutant mouse model. In the new project, we plan to build on our recent findings and perform further in-depth analysis of the role of CaBP2 and 1 in the regulation of mouse hair cell Ca2+ influx and exocytosis, and sound encoding in the cochlea. As biochemical data on CaBP2 is still missing, we will first study the secondary structure of purified wild-type and mutant CaBP2 using CD spectroscopy and analyze their Ca2+ binding properties by microscale thermophoresis and fluorimetric studies. Next, using recently generated knock-out mouse models and virus-mediated gene transfer into the embryonic otocyst, we will study the regulation of CaV1.3 channels by wild-type and selected mutant CaBP2s, related to pathologic mutations found in humans. The hearing of (single and double) knock-out mice (CaBP2-/- and CaBP1/2-/-) expressing the CaBP2 constructs will be investigated in detail on the systems and the cellular level. We will perform IHC patch-clamp recordings of Ca2+ and Ba2+ currents to investigate current inactivation kinetics, measure changes in membrane capacitance to detect differences in IHC exocytosis, and perform Ca2+ imaging to examine presynaptic Ca2+ signals. For systems physiology and single auditory nerve fiber recordings in rescued CaBP1/2 knock-outs expressing various CaBP2 constructs, we will collaborate within the consortium (Dr. Nicola Strenzke), to examine sound encoding. Finally, in case of calmodulin, distinct roles of its N- and C-terminal lobes have been suggested; but data for CaBPs in this context are still lacking. The available CaBP2 mutant constructs will now allow us to separately assess the function of each of the two CaBP2 lobes in order to properly address this question.

电压门控CaV1.3（L型）Ca 2+通道介导听觉内毛细胞（MHC）带状突触处的Ca 2+内流和胞吐，并且是听力所需的。这些通道经历由钙调蛋白介导的进入的Ca 2+离子的负反馈调节（Ca 2+依赖性失活）。钙调蛋白的作用被钙结合蛋白（Ca 2 + binding proteins，CaBP）阻止，CaBP与钙调蛋白一起属于含EF-手基序的蛋白质大家族。先前，已经证明了CaBP 1、2、4和5在小鼠耳蜗IHC中的表达。尽管CaBP 4的遗传缺失仅导致IHC Ca 2+内流的微小改变，使听力不受干扰，但最近已确定CaBP 2的突变是人类患者中常染色体隐性耳聋（DFNB 93）的非综合征形式的基础。一项正在进行的研究目前正在突变小鼠模型中调查这种听力障碍的细胞疾病机制。在新项目中，我们计划在我们最近的研究结果的基础上，进一步深入分析CaBP 2和1在调节小鼠毛细胞Ca 2+内流和胞吐以及耳蜗中声音编码中的作用。由于CaBP 2的生化数据仍然缺失，我们将首先使用CD光谱研究纯化的野生型和突变型CaBP 2的二级结构，并通过微尺度热泳和荧光研究分析其Ca 2+结合特性。接下来，使用最近产生的基因敲除小鼠模型和病毒介导的基因转移到胚胎耳囊中，我们将研究野生型和选定的突变CaBP 2s对CaV1.3通道的调节，这与人类中发现的病理突变有关。将在系统和细胞水平上详细研究表达CaBP 2构建体的（单和双）敲除小鼠（CaBP 2-/-和CaBP 1/2-/-）的听力。我们将进行Ca 2+和Ba 2+电流的IHC膜片钳记录以研究电流失活动力学，测量膜电容的变化以检测IHC胞吐的差异，并进行Ca 2+成像以检查突触前Ca 2+信号。对于系统生理学和单个听觉神经纤维记录在获救的CaBP 1/2敲除表达各种CaBP 2结构，我们将在财团（尼古拉Strenzke博士）内合作，以检查声音编码。最后，在钙调蛋白的情况下，其N-和C-末端叶的不同作用已被建议，但在这种情况下的CaBPs的数据仍然缺乏。现有的CaBP 2突变体构建体现在将允许我们分别评估两个CaBP 2叶中每一个的功能，以正确解决这个问题。