MECHANOTRANSDUCTION IN BARORECEPTOR NEURONS AND DROSOPHILA SENSORY NEURONS

动脉压力感受器是局限于颈动脉的感觉末梢 窦和主动脉弓，但具有强大的调节影响。他们 动脉压信号进入神经体液调节 心血管结构和体内平衡。尽管有大量的 关于压力感受器（BR）神经激活的决定因素的信息， 机械电的分子、细胞和功能决定因素 在它们的感觉神经末梢的传导在很大程度上是未知的。项目 本PPG的第四部分讨论了机械感觉的神经生物学。这 四年前启动的项目（四.A.1），重点是： 鉴定结状神经节中的BR神经元; 2）将它们培养在 体外; 3）建立了一个定量系统，为他们的机械 激活;以及4）通过测量其响应的定义 全细胞膜电流，单通道开放[Ca 2 +]i.一 通过测量来寻找细胞机制的功能相关性 颈动脉窦神经活动的影响。基于非常令人兴奋的 初步研究，我们现在建议测试的假设， ENaC/退化蛋白家族蛋白代表离子通道，负责 BR神经元中的机械电转导。这一假设获得了 从触摸和身体接触之间的遗传联系的发现中， 在C. Elegans和机械激活的证据 ENaC/简并蛋白通道。在初步研究中，我们发现：（1） ENAC/退化蛋白的亚基，γ ENaC; β ENaC和BNC 1（最近 克隆自脑），在动脉瘤的主动脉弓外膜中表达。 BR神经末梢和BR神经元所在的结状神经节， 2）BNC 1a（转染到卵母细胞中）的活性是 被Gd 3+（一种已知的机械感觉通道的阻断剂）阻断;和3） 在机械刺激期间BR神经元中[Ca 2 +]i的升高被以下物质阻断： 阿米洛利，一种已知的ENaC/变性蛋白通道阻滞剂。我们的目标是： 第一，鉴定ENaC/简并蛋白ENaC/简并蛋白通道;第三， 试图破坏BR对机械刺激的反应， 细胞，器官和整个动物水平的转染显性- ENaC/Degenerin家族阴性。 该项目的独特之处在于将两个领先的 实验室，具有自主控制神经生物学的专业知识（Abboud） 和离子通道的分子生物学（威尔士）。与添加的 支持成像、转基因动物和基因 转移，成功追求这个问题的承诺是 确定。

The arterial baroreceptors are sensory endings restricted to the carotid sinus and aortic arch but with powerful regulatory influences. They transduce arterial pressure signals into neurohumoral regulation of cardiovascular structure and homeostasis. Despite a large amount of information on the determinants of activation of baroreceptor (BR) nerves, the molecular, cellular and functional determinants of mechanoelectrical transduction at their sensory nerve endings are largely unknown. Project IV of this PPG addresses the neurobiology of mechanosensation. This Project (IV.A.1) which was initiated four years ago, as focused on 1) the identification of BR neurons in the nodose ganglion; 2) culturing them in vitro; 3) the development of a quantitative system for their mechanical activation; and 4) the definition of their responses with measurements of whole cell membrane currents, single-channel openings [Ca2+]i. A functional correlate of the cellular mechanisms was sought by measurements of carotid sinus nerve activity in vivo. Based on very exciting preliminary studies, we now propose to test the hypothesis that the ENaC/degenerin family of proteins represent ionic channel responsible for mechanoelectrical transduction in BR neurons. This hypothesis gains a strong rationale from the discovery of a genetic link between touch sensation in C. Elegans and the evidence of mechanical activation of ENaC/degenerin channels. In preliminary studies we found that 1) the subunits of ENAC/degenerin, gamma ENaC; beta ENaC and BNC1 (recently cloned from brain), are expressed in the aortic arch adventitia in the nodose ganglia where the BR nerve terminals and BR neurons are located, respectively; 2) the activity of BNC1a (transfected into oocytes) is blocked by Gd3+, a known blocker of mechanosensory channels; and 3) the rise in [Ca2+]i in BR neurons during mechanical stimulation is blocked by amiloride, a known blocker of the ENaC/degenerin channels. Our aims are: first, to identify ENaC/degenerin ENaC/degenerin channels; and third, to attempt to disrupt the BR responses to mechanical stimulation at the cellular, organ and whole animal level by transfection of dominant- negatives of ENaC/degenerin family. The uniqueness of the project is in the joining of two leading laboratories, with expertise in neurobiology of autonomic control (Abboud) and in the molecular biology of ion channels (Welsh). With the added support of excellent cores in imaging, transgenic animals, and gene transfer, the promise of successful pursuit of this problem is ascertained.