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

电压门控型CaV1.3(L型)钙通道介导听觉内毛细胞带状突触的钙内流和胞吐，是听力所必需的。这些通道受到钙调素介导的钙离子(钙依赖的失活)的负反馈调节。钙调素的作用被钙结合蛋白(CaBP)所阻止，钙结合蛋白与钙调素一起属于含EF-Hand-Motif蛋白的大家族。此前，CaBP1、2、4和5在小鼠耳蜗间充质细胞中的表达已被证明。虽然CaBP4的基因缺失只会导致IHC钙离子流入的微小变化，但听力没有受到影响，但最近发现CaBP2基因突变是人类患者中一种非综合征形式的常染色体隐性遗传性耳聋(DFNB93)的基础。目前正在进行的一项研究在突变小鼠模型中调查这种听力障碍的细胞疾病机制。在新的项目中，我们计划在我们最新发现的基础上，进一步深入分析CaBP2和1在调节小鼠毛细胞钙内流和胞吐以及耳蜗声编码中的作用。由于CaBP2的生化数据仍然缺乏，我们将首先利用CD光谱研究纯化的野生型和突变型CaBP2的二级结构，并通过微尺度的热电泳法和荧光法分析它们的钙结合特性。接下来，利用最近建立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，我们将研究与人类发现的病理突变相关的野生型和突变型CaBP2s对CaV1.3通道的调节。表达CaBP2结构的(单、双)基因敲除小鼠(CaBP2-/-和CaBP1/2-/-)的听力将在系统和细胞水平上进行详细的研究。我们将进行IHC膜片钳记录钙电流和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胞吐的差异，并进行钙成像以检查突触前钙信号。对于被拯救的表达各种CaBP2结构的CaBP1/2基因敲除基因中的系统生理学和单个听觉神经纤维记录，我们将在该联盟(Nicola Strenzke博士)内部合作，以检查声音编码。最后，在钙调蛋白的情况下，已经提出了其N-末端和C-末端叶的不同作用；但在这方面关于CaBP的数据仍然缺乏。可用的CaBP2突变结构现在将允许我们分别评估两个CaBP2叶的功能，以便适当地解决这个问题。

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.