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

电压门控CaV1.3 （l型）Ca2+通道在听觉内毛细胞（IHC）带突触介导Ca2+内流和胞外分泌，是听力所必需的。这些通道经历钙调蛋白介导的钙离子（Ca2+依赖性失活）的负反馈调节。钙离子结合蛋白（cabp）与钙调蛋白一起属于含有ef -hand基元的蛋白大家族，钙离子结合蛋白（cabp）可以阻止钙调蛋白的作用。先前，CaBP1、2、4和5在小鼠耳蜗ihc中的表达已被证实。虽然CaBP4基因缺失仅引起IHC Ca2+内流的轻微改变，而听力不受干扰，但CaBP2突变最近被确定为人类患者常染色体隐性耳聋（DFNB93）的非综合征形式的基础。目前，一项正在进行的研究在突变小鼠模型中调查了这种听力障碍的细胞疾病机制。在新项目中，我们计划以我们最近的发现为基础，进一步深入分析CaBP2和1在调节小鼠毛细胞Ca2+内流和胞外分泌以及耳蜗声音编码中的作用。由于CaBP2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CaBP2的二级结构，并通过微尺度热泳术和荧光学研究分析它们的Ca2+结合特性。接下来，我们将利用最近建立的敲除小鼠模型和病毒介导的基因转移到胚胎耳囊肿中，研究野生型和选择突变的CaBP2s对CaV1.3通道的调控，这与人类发现的病理突变有关。我们将在系统和细胞水平上详细研究表达CaBP2结构的（单敲除和双敲除）小鼠（CaBP2-/-和CaBP1/2-/-）的听力。我们将进行IHC膜片钳记录Ca2+和Ba2+电流以研究电流失活动力学，测量膜电容的变化以检测IHC胞外分泌的差异，并进行Ca2+成像以检查突触前Ca2+信号。对于表达多种CaBP2结构的获救CaBP1/2敲除者的系统生理学和单听神经纤维记录，我们将与Nicola Strenzke博士合作，研究声音编码。最后，在钙调素的情况下，其N端和c端叶的不同作用已被提出；但这方面的cabp数据仍然缺乏。现有的CaBP2突变体结构现在将允许我们分别评估两个CaBP2叶的功能，以便正确地解决这个问题。

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.