Inducible Expression of Exogenous Potassium Channels in Hair Cells

描述（由申请人提供）：本R21研究项目的目标是开发和表征一种新的小鼠模型，该模型将用于沉默听觉和前庭毛细胞活动。我们将产生一个转基因小鼠系，允许诱导，毛细胞表达TREK1钾通道；也称为KCNK2或K2P2.1通道。由于TREK1通道在静止状态下是开放的，并且不是电压门控的，它们的表达将引入一个电分流到毛细胞膜上，这将有效地“钳住”毛细胞，使其接近钾平衡电位，约为-80 mV。这个简单的模型将使毛细胞电沉默，不能执行电压依赖的功能。该小鼠模型将被用于研究听觉和前庭系统中依赖于毛细胞膜电位的许多功能，这是其他技术无法实现的。我们计划利用该模型来研究外毛细胞膜电位在耳蜗放大中的作用，毛细胞/第8神经传入连接处自发活动在突触发生中的作用，刺激诱发活动在听觉和前庭系统发育和成熟中的作用。我们还将使用该系统来研究与年龄相关的听力和平衡功能障碍小鼠模型以及功能恢复过程中的经验依赖可塑性。为了生成我们的新小鼠模型，我们将使用一种细菌Lac操作符/抑制因子系统，该系统已经过修改，可用于小鼠（Cronin et al., 2001）。我们将在Myosin7a启动子中引入Lac算子。Myo7a启动子在动物的整个生命周期中都是活跃的，从胚胎第12天开始（Boeda et al., 2001）。修饰后的Myo7a启动子在Lac I蛋白存在下将失去活性。仅仅通过添加诱导剂，一种无毒的乳糖类似物（IPTG），抑制将被缓解，TREK1的表达将开始。因此，使用一个简单的化学开关，我们将能够在老鼠的生命周期的任何时候打开或关闭毛细胞的活动。我们认为这项工作对于理解受体潜能对许多关键毛细胞功能的贡献将是重要的。这种新型诱导系统的发展也将提供一个强大的工具，可以修改来控制毛细胞表达的任何外源基因感兴趣。因此，这个新工具将使我们能够解决关于听觉和前庭功能基础生物学的几个长期存在的问题，也可能发展为研究听力和平衡障碍的新策略，这些疾病共同影响着全球2.5亿人。

DESCRIPTION (provided by applicant): The goal of this R21 research project is to develop and characterize a new mouse model that will be used to silence auditory and vestibular hair cell activity. We will generate a transgenic mouse line that allows for inducible, hair-cell expression of TREK1 potassium channels; also know as KCNK2 or K2P2.1 channels. Because TREK1 channels are open at rest and are not voltage-gated their expression will introduce an electrical shunt into the hair cell membrane which will effectively "clamp" the hair cell near the potassium equilibrium potential, about -80 mV. This simple model will render hair cells electrically silent, unable to perform voltage-dependent functions. The mouse model will be used to investigate, in a manner not available by other techniques, a number of functions in the auditory and vestibular systems that depend on the hair cell membrane potential. We plan to use the model to examine the role of the outer hair cell membrane potential in cochlear amplification, the role of spontaneous activity in synaptogenesis at the hair cell / 8th nerve afferent junction, the role of stimulus-evoked activity in the development and maturation of the auditory and vestibular systems. We will also use this system to investigate experience-dependent plasticity in a mouse model of age- related hearing and balance dysfunction, as well as during recovery of function. To generate our novel mouse model we will use a form of the bacterial Lac operator / repressor system that has been modified for use in mice (Cronin et al., 2001). We will introduce Lac operators into the Myosin7a promoter. The Myo7a promoter is active in hair cells throughout the animal's lifespan beginning as early as embryonic day 12 (Boeda et al., 2001). The modified Myo7a promoter will be inactive in the presence of Lac I protein. Simply by addition of the inducer, a nontoxic lactose analog, (IPTG), repression will be relieved and expression of TREK1 will commence. Therefore, using a simple chemical switch we will be able to turn on or off hair cell activity at anytime point during the lifespan of the mouse. We feel this work will be important for understanding the contribution of the receptor potential to a number of critical hair-cell functions. Development of this novel inducible system will also provide a powerful tool that can be modified to control hair-cell expression for any exogenous gene of interest. As such, this new tool will allow us to address several long standing questions regarding the basic biology of auditory and vestibular function and may also be developed into novel strategies for investigating hearing and balance disorders, which collectively affect >250 million people worldwide.