CFTR: Neuronal roles for a multifunctional transporter

神经元依靠它们的电活动来产生信号并与其他神经元进行交流。就像手电筒里的电池一样，离子梯度为神经元提供能量，使信息得以传递。在这里，我们研究了囊性纤维化跨膜调节剂（CFTR）的功能，CFTR是一种氯离子通道，当发生突变时，会导致囊性纤维化。由于这种与疾病的联系，CFTR在产生囊性纤维化危及生命的系统（如呼吸和消化系统）的背景下得到了大量研究。相比之下，人们对CFTR在大脑中的功能知之甚少，在大脑中也发现了CFTR。已知CFTR在神经元中的作用之一是调节氯离子梯度。这个新项目将深入研究CFTR在神经元中的调控及其独特功能。该项目的主要目标之一是确定与CFTR相互作用的神经元特异性蛋白质。本研究将提供CFTR在大脑中的作用，并为进一步研究这一多功能离子通道提供神经蛋白相互作用信息的支架。一项名为GeauxNeuro的中学神经科学拓展项目已经实施，该项目为路易斯安那州立大学的神经科学研究生提供了拓展机会，并将神经科学带给路易斯安那州公立学校的学生。尽管CFTR在脑和脊髓神经元中的表达已被证实，但这种Cl-通道在神经元功能中的作用尚不清楚。神经元中CFTR的表达逐渐被认识到，然而，PI只能确定不超过15项直接研究CFTR在神经元中的功能。本研究的重点是CFTR在神经元胞浆Cl-水平调控和突触功能调控中的作用。在Aim 1中，将阐明CFTR的定位和监管。由于生理实验表明CFTR在酸性细胞器的膜中表达，因此CFTR的亚细胞定位将在电子显微镜水平上使用免疫细胞化学进行检测。电生理和分子生物学实验旨在发现CFTR与no依赖的内部Cl-释放之间的机制联系。在Aim 2中，将使用类似的方法来研究CFTR和两个已建立的相互作用物（anoctamin 1和syntaxin 1A）的功能意义，以确定这些相互作用在突触前和突触后操作中的作用。众所周知，CFTR与其他蛋白质具有高度细胞类型特异性的相互作用，因此，Aim 3定义了一种基于免疫沉淀-质谱的策略，用于鉴定这种多功能蛋白质的新型神经元结合伙伴。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Neurons rely on their electrical activity to generate signals and to communicate with other neurons. Like batteries in a flashlight, ion gradients power neurons and enable the transmission of information. Here, we study the function of cystic fibrosis transmembrane regulator (CFTR), a chloride channel that when mutated, causes the disease cystic fibrosis. Because of this link to disease, CFTR has been heavily studied in the context of systems that produce the life-threatening effects of cystic fibrosis such as the respiratory and digestive systems. In contrast, little is known about the function of CFTR in the brain where it is also found. It is known that one role of CFTR in neurons is to regulate the chloride ion gradient. This new project will take a deep dive into the regulation and unique functions of CFTR in neurons. One of the major objectives of this project is to identify the neuron-specific proteins with which CFTR interacts. This research will provide insight into the role of CFTR in the brain and a scaffold of neuronal protein interaction information which will form the basis of further studies into this multifunctional ion channel. A middle school neuroscience outreach program called GeauxNeuro has been implemented that provides a diverse group of neuroscience graduates students at LSU with outreach opportunities and brings neuroscience to public school students in Louisiana. Although CFTR expression has been demonstrated for neurons in the brain and spinal cord, the role of this Cl- channel in neuronal function is poorly understood. The expression of CFTR in neurons is becoming recognized, however, the PI could identify no more than 15 studies that directly address CFTR function in neurons. This proposal focuses on the role of CFTR in the management of neuronal cytosolic Cl- levels and in regulating synaptic function. In Aim 1 both the localization and regulation of CFTR will be elucidated. Subcellular localization of CFTR will be examined using immunocytochemistry at the electron microscope level because physiological experiments suggest that CFTR is expressed in the membranes of acidic organelles. Electrophysiological and molecular biology experiments are designed to discover the mechanistic links between CFTR and an NO-dependent release of internal Cl-. In Aim 2, similar methods will be used to investigate the functional significance of CFTR and two established interactors (anoctamin 1 and syntaxin 1A) to determine the role these interactions play in pre- and postsynaptic operations. CFTR is well known to have highly cell-type specific interactions with other proteins and as such, Aim 3 defines an immunoprecipitation-mass spectroscopy-based strategy for identifying novel neuronal binding partners for this multi-functional protein.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.