Development of endocytosis in cochlear inner hair cells

哺乳动物内毛细胞(IHC)不知疲倦地将机械声刺激转化为分级受体电位和分级递质释放，在很大的动态范围内具有良好的时间精度。在听力开始之前，IHC经历了一个发育过程，在这个过程中它们产生了钙离子驱动的动作电位。在听前和成熟的IHC之间，递质释放在许多方面都不同，例如：i)触发类型(Ca~(2+)动作电位与分级受体电位)，ii)Ca~(2+)效率，III)关键分子和iv)突触的形态。虽然囊泡释放的功能和成熟度在IHC中得到了很好的研究，但这不是先前从质膜插入的囊泡膜的内吞作用的情况。初步数据有力地表明，内吞作用经历了一个成熟的过程，与胞吐作用的发育变化相平行。在这个项目中，我的目标是比较早熟和成熟的IHC的内吞作用。其具体目的是：1)表征内吞作用的动力学和对钙的依赖性；2)确定内吞机制的分子组成；3)分析内吞蛋白的作用。内吞作用将在膜电容改变时使用膜片钳技术进行测量。我们将通过转录分析在mRNA水平上鉴定内吞机制的成分，并通过光学显微镜和透射电子显微镜进行免疫标记来鉴定蛋白质水平上的内吞机制的成分。最后，我们将通过阻断内吞蛋白的功能，并通过膜电容记录和使用固定膜染料的膜摄取成像来研究内吞作用的结果，来阐明内吞蛋白在IHC中的作用。在这里获得的知识对于理解IHC的最终成熟以及IHC如何不知疲倦地将声音信息传递到听觉通路是必不可少的。这项研究将进一步作为未来项目的基础，该项目涉及内吞蛋白突变小鼠模型，以了解人类耳聋。

Mammalian inner hair cells (IHC) indefatigably transduce mechanical sound stimuli into graded receptor potentials and graded transmitter release with excellent temporal precision over a wide dynamic range. Before the onset of hearing, IHCs undergo a developmental program during which they generate Ca2+-driven action potentials. Transmitter release differs in many regards between pre-hearing and mature IHCs, such as i) the kind of trigger (Ca2+ action potentials vs. graded receptor potentials), ii) Ca2+ efficiency, iii) crucial molecules and iv) morphology of the synapse. Whereas function and maturation of vesicle release is well studied in IHCs, this is not the case for endocytosis of previously inserted vesicle membrane from the plasma membrane. Preliminary data strongly suggest that endocytosis undergoes a process of maturation that parallels the developmental modifications of exocytosis. In this project I aim to characterise endocytosis in pre-mature compared to mature IHCs. The specific aims are: 1) to characterise the kinetics and Ca2+ dependence of endocytosis; 2) to identify the molecular components of the endocytic machinery; 3) to analyse the role of endocytic proteins. Endocytosis will be measured as membrane capacitance changes using the patch clamp technique. We will identify components of the endocytic machinery on mRNA level by transcript analysis and on protein level by immunolabelling using light microscopy and transmission electron microscopy. Finally, we will elucidate the role of endocytic proteins in IHCs by blocking their function and studying the resulting effect on endocytosis via membrane capacitance recordings and imaging of membrane uptake using a fixable membrane dye. The knowledge gained here is essential to understand final maturation of the IHC and how IHCs indefatigably transfer sound information to the auditory pathway. This study will further serve as a basis for future projects involving mutant mouse models of endocytic proteins for understanding human deafness.