Cellular/Molecular Mechanisms of Respiratory Neuronal Chemosensitivity

描述(申请人提供)：中枢呼吸化学接收是一种众所周知的和极其敏感的动态平衡机制，大脑通过调节呼吸来维持生理上适当的pH和PCO2水平。中枢化学接收功能障碍与各种中枢性呼吸紊乱有关(例如，婴儿猝死、中枢性先天性低通气量)，强调了这一系统的重要性。要从根本上理解这个重要的感觉反馈系统，就需要识别相关的传感器--包括涉及的神经元和它们的分子探测器。在这一点上，延髓反斜方核(RTN)中离散的神经元群体满足化学感受器神经元的关键标准。然而，关键问题仍然存在。目前尚不清楚pH感知是否是这些神经元的固有属性，这是继续被认为是化学传感器所必需的，RTN神经元pH敏感性的细胞/分子基础也尚未阐明。为了解决这些基本问题，本拨款申请中提出的研究将复杂的分子和体外电生理学技术应用于一种新的小鼠品系，该品系在对化疗敏感的RTN神经元中选择性表达绿色荧光蛋白(GFP)。支持特定目标1的假设是，RTN神经元具有内在的化学敏感性，不同类型RTN神经元的特征pH反应反映了背景通道的不同互补。在一个子目标中，实验提供了脑干切片中记录的RTN神经元对pH敏感的本底K电流和TTX抵抗的泄漏Na电流的详细特征，并通过使用慢病毒介导的shRNA敲除来测试泄漏Na电流是否由NALCN通道携带。在第二个子目标中，在分离的细胞系统中记录表达GFP的RTN神经元，以确定pH敏感性是否是RTN神经元的固有属性。驱动特定目的2的假说是，负责rtn神经元化疗敏感性的K通道(S)在静息膜电位上具有显着的结构性活性，它们对pH固有地敏感，或者它们是pH激活的G蛋白偶联受体的下游效应器。三个子目标测试了在RTN神经元中表达的不同候选pH传感器的参与；其中包括电压门控(KV)和两孔结构域(K2P)通道家族的不同K通道，以及质子激活的G蛋白偶联受体。为此，当候选分子传感器的功能表达被基因敲除或慢病毒介导的显性负向或shRNA结构的表达中断后，在表达GFP的RTN神经元中获得记录。拟议的研究提供了关于这一重要的动态平衡调节系统的关键信息。识别新的分子底物，作为中枢呼吸化学接收的基础，可以为呼吸障碍的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Central respiratory chemoreception is a well known and exquisitely sensitive homeostatic mechanism by which the brain maintains physiologically appropriate levels of pH and PCO2 via regulation of breathing. Dysfunction of central chemoreception is implicated in various central disorders of breathing (e.g., sudden infant death, central congenital hypoventilation), underscoring the critical importance of this system. A fundamental understanding of this important sensory feedback system demands identification of the relevant sensors - both the neurons involved and their molecular detectors. In this regard, a discrete population of neurons in the retro-trapezoid nucleus (RTN) of the medulla oblongata fulfills key criteria expected for chemoreceptor neurons. However, key questions remain. It is not known if pH sensing is an intrinsic property of those neurons, as required for continued consideration as chemosensors, and the cellular/molecular basis for RTN neuronal pH sensitivity has not been elucidated. In order to address these fundamental issues, research proposed in this grant application applies sophisticated molecular and in vitro electrophysiological techniques in the context of a new line of mice that express green fluorescent protein (GFP) selectively in chemosensitive RTN neurons. The hypothesis underpinning Specific Aim 1 is that RTN neurons are intrinsically chemosensitive, and that characteristic pH responses for different types of RTN neurons reflect distinct complements of background channels. In one sub-aim, experiments provide detailed characterization of pH-sensitive background K+ current and TTX-resistant leak Na+ current in RTN neurons recorded in brainstem slices, and they test if the leak Na+ current is carried by NALCN channels by using lentiviral-mediated shRNA knockdown. In a second sub-aim, GFP-expressing RTN neurons are recorded in a dissociated cell system to test definitively if pH sensitivity is an intrinsic property of RTN neurons. The hypothesis driving Specific Aim 2 is that the K+ channel(s) responsible for RTN neuronal chemosensitivity have significant constitutive activity at resting membrane potentials and they are intrinsically sensitive to pH or they are downstream effectors for pH-activated G protein-coupled receptors. Three sub-aims test involvement of different candidate pH-sensors that are expressed in RTN neurons; these include distinct K+ channels of the voltage-gated (KV) and two-pore-domain (K2P) channel family, as well as proton-activated G protein-coupled receptors. For this aim, recordings are obtained in GFP-expressing RTN neurons after functional expression of candidate molecular sensors is disrupted by genetic knockout or lentiviral-mediated expression of dominant-negative or shRNA constructs. The proposed studies provide critical information regarding this important homeostatic regulatory system. Identification of novel molecular substrates that underlie central respiratory chemoreception could provide new targets for therapeutic intervention in disorders of breathing.