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

描述（由申请人提供）：目前全球约有3400万人感染人类免疫缺陷病毒（HIV）。胃肠道（GI）是HIV病毒的主要靶点和储存库，其含有大量淋巴细胞，并且与外周血相比，在急性和慢性HIV感染期间具有更高水平的CD 4 T细胞耗竭。肠神经系统调节GI过程，如运动和消化。这些过程在HIV感染患者中受到显著影响。HIV病毒不会感染神经元。大多数HIV效应不是通过病毒的裂解繁殖而是通过病毒蛋白实现的。HIV-1达特，一种长度在86至101个氨基酸之间的调节蛋白，是这样的蛋白质之一。达特由受感染的细胞释放，并能够穿透和调节神经元功能。此外，达特在逃避潜伏期方面发挥着重要作用，这是获得性免疫缺陷综合症（艾滋病）发病机制的标志。最近的实验和临床研究表明，肠神经系统在HIV感染期间受到影响，从而导致HIV介导的GI神经胃肠病。大量关于达特对神经元作用的研究是在中枢神经系统（CNS）神经元上进行的。在中枢神经系统中，达特已被证明能增加神经元的兴奋性，但其发生的机制仍不清楚。基于我们的初步数据，我们假设达特通过调节钠通道特性增加肠神经元兴奋性。我们将测试的假设，达特增加神经元的兴奋性在体外系统和动物模型（多西环素诱导的达特转基因小鼠）使用全细胞膜片钳研究从成年小鼠回肠的单个分离的神经元。然后，我们将通过检查达特对钠通道的影响来确定达特增强肠神经元兴奋性的机制，所述钠通道在神经元兴奋性中起重要作用。我们将建立在我们的初步数据上，这些数据表明达特（1）增加钠电流密度，（2）使玻尔兹曼激活曲线向左移动，（3）增加钠通道的Nav1.7和Nav1.8亚型的转录。我们将确定激活曲线的这种快速移动是达特的急性、直接还是变构效应。我们将使用电压钳实验和双脉冲失活方案对肠神经元用达特处理长达30分钟，以确定它是否是一个急性效应。我们将使用免疫共沉淀来确定它是否是达特的直接作用，并使用IP/免疫印迹技术来评估该作用是否是间接和/或翻译后修饰。然后，我们将阐明达特转录调节Nav1.7和Nav1.8亚型的机制，以及Nav1.7和Nav1.8转录的增加是否被翻译成蛋白质，以及达特是否也调节这些亚型的翻译。

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.