MicroRNA permeability of connexin mutants related to genetic disease

项目总结/摘要 缝隙连接对于正常组织生理学是必不可少的，为细胞提供了直接的细胞间通路， 细胞信号和脉冲传导。它们的亚基蛋白（连接蛋白）基因的突变已经被发现。 与许多人类疾病有关。例如，连接蛋白40（Cx40）的突变与 房颤和其他心律失常三个Cx40突变（A96 S，M163 V和G38 D）与 先前已经显示出心房纤颤患者保留形成传导性间隙连接通道的能力 体外我们最近的出版物表明，三个中的两个具有相同的幺正电导， 电压依赖性行为的野生型Cx40，而第三个具有显着更高的单一 电导所有三种突变体相对于野生型通道具有改变的渗透性特征。一个 如今越来越多的证据表明microRNA在发病机制中的重要作用， 心脏病的发展。这些数据提示了另一种尚未证实的可能性： 突变体可能通过改变微RNA的渗透性来影响心脏传导和起搏。 心肌本身靶向特异性膜K+通道IK 1（KCNJ 2/Kir2.1）和IKr（HERG）， 参与传导和起搏活动。最近有报道称，microRNA miR-26 调节IK 1的表达，并在房颤中下调。miR-212也被证明影响IK 1 IKr的表达受miR-133的影响。 在这项研究中，我们将测试起搏器的活性是否可以通过连接蛋白的通透性来调节， microRNAs/siRNAs，反过来调节特定膜通道的基因表达， 起搏器样活动。在目标1中，我们将确定由以下形成的间隙连接通道的渗透性： Cx40至miR-26、miR-212、miR-133以及IK 1和IKr特异性siRNA的疾病相关突变， 同时监测细胞的结电导和细胞间通量， microRNA/siRNA。在Aim 2中，我们将使用双细胞起搏器模型（由一个细胞组成的异源对）。 源细胞和心肌细胞），以探索通过细胞内递送microRNA对起搏器活性的影响 以及靶向IK 1和IKr的siRNA。此后，在Aim 3中，我们将使用我们的动态钳位来分析这些结果 为了向/从单个细胞添加和减去与起搏器活动相关联的相关电流， 耦合对，或两个未耦合的电池以电子方式配对。 在这项拟议的研究中，使用细胞生物物理学方法，我们试图确定间隙的渗透性是否 连接通道可能是动态基因表达过程中的重要组成部分， 影响正常生理和疾病状态下的细胞和组织功能。

Project Summary/Abstract Gap junctions are essential to normal tissue physiology, providing a direct intercellular pathway for cell to cell signaling and impulse conduction. Mutations in the genes of their subunit protein (connexins) have been linked to a number of human diseases. For instance, mutations in connexin40 (Cx40) have been associated with atrial fibrillation and other arrhythmias. Three Cx40 mutations (A96S, M163V and G38D) that correlate with atrial fibrillation have been previously shown to retain the ability to form conductive gap junction channels in vitro. Our recent publication demonstrates that two of the three have the same unitary conductance and voltage dependent behaviors as wild-type Cx40, while the third one has a significantly higher unitary conductance. All three mutants have altered permeability characteristics relative to wild-type channels. An increasing amount of evidence today indicates the important role of microRNAs in the pathogenesis and development of heart diseases. The data suggest another and as of yet untested possibility; these connexin mutants might affect cardiac conduction and pacing via altered permeability to microRNAs within the myocardium, which themselves target specific membrane K+ channels, IK1 (KCNJ2/Kir2.1) and IKr (HERG), that participate in conduction and pacemaking activities. It was recently reported that the microRNA miR-26 regulates IK1 expression and is down regulated in atrial fibrillation. miR-212 has also been shown to affect IK1 expression, whereas IKr expression is affected by miR-133. In this proposed study we will test if pacemaker activity can be modulated by connexin permeability to microRNAs/siRNAs that in turn regulate gene expression of specific membrane channels associated with pacemaker-like activity. In Aim 1 we will determine the permeability of gap junction channels formed by disease-linked mutations in Cx40 to miR-26, miR-212, miR-133, and siRNAs specific to IK1 and IKr, respectively, while simultaneously monitoring junctional conductance and cell to cell flux of microRNAs/siRNAs. In Aim 2 we will use a two-cell pacemaker model (a heterologous pair comprised of a source cell and a cardiac myocyte) to explore effects on pacemaker activity, by cellularly delivering microRNAs and siRNAs that target IK1 and IKr. Thereafter, in Aim3, we will analyze these results using our dynamic clamp model in order to add and subtract relevant currents associated with pacemaker activity to/from a single cell, a coupled pair, or two uncoupled cells paired electronically. In this proposed study, using a cellular biophysical approach, we seek to define if the permeability of gap junction channels might be an essential component in a process of dynamic gene expression that ultimately affects cellular and tissue functions in normal physiology and disease states.