Modulation of sodium channels by HIV-1Tat in enteric neurons

描述(申请人提供)：目前全球约有3400万人携带人类免疫缺陷病毒(HIV)。胃肠道(GI)是HIV病毒的主要目标和储存库，其中含有大量的淋巴细胞，在急性和慢性HIV感染期间，与外周血相比，CD4T细胞的耗竭水平更高。肠道神经系统调节胃肠道运动和消化等过程。这些过程在感染艾滋病毒的患者中受到很大影响。HIV病毒不会感染神经元。HIV的大部分作用不是通过病毒的裂解繁殖实现的，而是通过病毒蛋白实现的。HIV-1Tat是一种长度在86到101个氨基酸之间的调节蛋白，就是这样的蛋白质之一。TAT由受感染的细胞释放，能够穿透并调节神经元功能。此外，TAT在逃避潜伏期方面发挥着重要作用，潜伏期是获得性免疫缺陷综合征(AIDS)发病机制的一个标志。最近的实验和临床研究表明，在HIV感染过程中，肠道神经系统受到影响，从而导致HIV介导的胃肠神经胃肠病。大部分关于TAT对神经元影响的研究都是在中枢神经系统(CNS)神经元上进行的。在中枢神经系统中，TAT已被证明可以增加神经元的兴奋性，但其发生的机制仍不清楚。根据我们的初步数据，我们假设TAT通过调节钠通道特性来增加肠神经兴奋性。我们将在体外系统和动物模型(多西环素诱导的TAT转基因小鼠)中使用成年小鼠回肠单个分离神经元的全细胞膜片钳研究来检验TAT增加神经元兴奋性的假设。然后，我们将通过检测TAT对钠通道的影响来确定TAT增强肠神经兴奋性的机制，钠通道在神经元兴奋性中起着重要作用。我们将以我们的初步数据为基础，这些数据表明TAT(1)增加了钠电流密度，(2)使Boltzmann的激活曲线左移，(3)增加了钠通道的Nav1.7和Nav1.8亚型的转录。我们将确定激活曲线的这种左移是TAT的急性、直接或变构效应。我们将使用电压钳实验和双脉冲灭活方案对肠神经元进行TAT长达30分钟的处理，以确定这是否为急性影响。我们将使用免疫共沉淀来确定它是否是TAT的直接影响，以及IP/免疫印迹技术来评估这种影响是否是间接和/或翻译后修饰。然后，我们将阐明TAT转录调控Nav1.7和Nav1.8亚型的机制，以及Nav1.7和Nav1.8转录的增加是否翻译成蛋白质，以及TAT是否也调控这些亚型的翻译。

DESCRIPTION (provided by applicant): About 34 million people are currently living with human immunodefiency virus (HIV) worldwide. The gastrointestinal (GI) tract is a major target and reservoir of the HIV virus containing a huge number of lymphocytes and having a higher level of CD4 T cell depletion during acute and chronic HIV infection compared to peripheral blood. The enteric nervous system regulates GI processes such as motility,and digestion. These processes are significantly affected in HIV infected patients. The HIV virus does not infect neurons. The majority of HIV effects are not achieved by lytic propagation of the virus but by viral proteins. HIV-1 Tat, a regulatory protein between 86 to 101 amino acids in length is one of such proteins. Tat is released by infected cells and is able to penetrate and modulate neuronal function. Additionally, Tat plays an important role in the escape from latency which is a hallmark of acquired immunodeficiency syndrome (AIDS) pathogenesis. Recent experimental and clinical studies suggest that the enteric nervous system is affected during HIV infection thereby contributing to HIV mediated GI neurogastroenteropathies. A bulk of the studies on the effects of Tat on neurons has been done on central nervous system (CNS) neurons. In the CNS, Tat has been shown to increase neuronal excitability but the mechanism by which this occurs is still not yet well understood. Based on our preliminary data, we hypothesize that Tat increases enteric neuronal excitability by modulating sodium channel properties. We will test the hypothesis that Tat increases neuronal excitability in both an in vitro system and an animal model (Doxycycline-inducible Tat transgenic mouse) using whole cell patch clamp studies in single isolated neurons from the adult mouse ileum. We will then determine the mechanism of enhanced enteric neuronal excitability by Tat by examining the effects of Tat on sodium channels which play an important role in neuronal excitability. We will build on our preliminary data which show that Tat (1) increases the sodium current density, (2) shifts the Boltzmann's activation curve to the left and (3) increases the transcription of Nav1.7 and Nav1.8 isoforms of the sodium channel. We will determine if this leftward shift in the activation curve is an acute, direct or allosteric effect of Tat. We will use voltage clamp experiments and a double pulse inactivation protocol on enteric neurons treated with Tat for up to 30min to determine if it is an acute effect. We will use co-immunoprecipitation to determine if it is a direct effect of Tat and a IP/Immunoblot technique to assess whether the effect is an indirect and/or post translational modification. We will then elucidate the mechanism by which Tat transcriptionally modulates the Nav1.7 and Nav1.8 isoforms, and whether this increase in Nav1.7 and Nav1.8 transcription is translated into proteins and if Tat is also modulating translation of these isoforms.