MicroRNA permeability of connexin mutants related to genetic disease

项目摘要/摘要 缝隙连接是正常组织生理所必需的，为细胞提供了直接的细胞间通路 细胞信号和脉冲传导。它们的亚单位蛋白(连接蛋白)的基因突变 与许多人类疾病有关。例如，连接蛋白40(Cx40)的突变与 伴有房颤和其他心律失常。三个相关的Cx40突变(A96S、M163V和G38D) 先前已经证明，房颤患者保留了形成传导缝隙连接通道的能力 在试管中。我们最近发表的文章表明，三者中的两个具有相同的么正电导和 电压依赖行为与野生型Cx40相同，而第三种具有显著更高的单一性 电导。这三个突变体都改变了相对于野生型通道的通透性特征。一个 越来越多的证据表明，microRNAs在肿瘤的发生和发展中发挥着重要作用。 心脏病的发展。这些数据表明了另一种尚未测试的可能性；这些连接蛋白 突变体可能通过改变心脏内对microRNA的通透性来影响心脏传导和起搏 心肌本身以膜K+通道Ik1(KCNJ2/Kir2.1)和Ikr(HERG)为靶标， 参与传导和起搏活动。最近有报道称，microRNA miR-26 调节IK1的表达，在房颤中下调。MIR-212也被证明影响IK1 而IKR的表达受miR-133的影响。 在这项拟议的研究中，我们将测试连接蛋白通透性是否可以调节起搏器的活动 MicroRNAs/siRNAs，反过来调节与以下相关的特定膜通道的基因表达 类似心脏起搏器的活动。在目标1中，我们将确定由以下形成的间隙结通道的渗透率 Cx40到miR-26、miR-212、miR-133的疾病相关突变，以及IK1和IKR特有的siRNA， 在同时监测结电导和细胞间通量的同时， MicroRNAs/siRNA。在目标2中，我们将使用两细胞起搏器模型(由 源细胞和心肌细胞)，以探索通过细胞传递microRNAs对起搏器活动的影响 以及针对IK1和IKR的siRNA。然后，在Aim3中，我们将使用我们的动态夹具分析这些结果 为了将与起搏器活动相关的相关电流加到单个细胞或从单个细胞减去起搏器活动， 耦合对，或两个未耦合的电子配对的电池。 在这项拟议的研究中，我们使用细胞生物物理方法，试图定义GAP的渗透性 连接通道可能是动态基因表达过程中的重要组成部分，最终 在正常生理和疾病状态下影响细胞和组织功能。

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.