Physiology and Biophysics of Cardiac Slo2.1 Channels

描述(由申请人提供)：大电导K通道由细胞内钠水平升高激活，于1984年首次在心脏中被描述。当时人们推测，这些通道传导的电流(IKNa)很可能只在严重缺血时激活，此后在很大程度上被忽略了。六年前，进行IKNa的频道被克隆并命名为Slo2.1。然而，目前对Slo2.1通道功能的结构基础知之甚少，包括通道激活的弱电压依赖性的分子基础，激活门的位置，以及细胞内钠结合与通道开放的耦合机制。我们的初步发现指出了Slo2.1的两个新特性。首先，通常的电压传感器(S1-S4段)对跨膜电压没有感知作用。其次，选择性过滤器，而不是S6束交叉，可以起到激活门的作用。在Aim1中，我们将使用定点突变、在非洲爪哇卵母细胞中的异源表达和电压钳技术来证实这些初步发现，并明确描述Slo2.1通道激活的分子基础。心脏骤停通常与室颤(VF)和缺血(VF/缺血)相结合，这两种情况都会促进[Na]i的升高。IKNa在VF/缺血期间的一个潜在但尚未被探索的作用是，它通过增加净外向电流和细胞外钾积累而导致抑制和兴奋性丧失。在长时间的室颤/缺血期间，心内膜和心外膜之间以及右和左心室(RV和LV)之间的激动率和兴奋性的梯度很大，这与除颤和休克后复苏的结果高度相关。然而，这些梯度的确切机制仍然是一个谜。我们的初步结果揭示了Slo 2.1表达的地区差异，这可能解释了电生理梯度。也有直接和间接的证据支持IKNa在缺血时钾外漏中的重要作用。不幸的是，对IKNa在缺血或室颤/缺血中的作用的了解非常有限。在目标2中，我们将检验一种假设，即在狗的心脏中，左心外膜是室颤/缺血期间第一个表现出不兴奋的区域，因为该区域的心肌细胞要么比左心内膜或右室/间隔积累更快的细胞质Na，要么表达更多密度的IKNa通道。当[Na]i短暂升高时，KNA通道在缺血、再灌流和快速心率时被激活。在目标3中，将在分离的犬心室肌细胞中检测在这些条件下KNA通道的激活。最后，还将在狗的整个心脏(目标2)和分离的心肌细胞(目标3)中研究IKNa和IKATP在这些现象中的相对贡献。这些研究将确定1)KNA通道激活的分子基础，2)这些通道在正常和病理生理条件下的作用，3)这些通道作为除颤和休克后复苏的调节器的潜在作用。 与公共健康相关：离子通道是膜结合的蛋白质，选择性地将特定的离子输送进和流出细胞。过多的钾离子通道在人体心脏中表达，是负责该器官正常泵功能的电活动的重要组成部分。一种类型的钾离子通道被称为slo2.1，只有当细胞内钠浓度升高到异常水平时，这些通道才会激活，就像在缺血期间可能发生的那样。心肌缺血是室性心动过速和纤颤的常见原因和后果，这两种形式的心律失常具有潜在的致命性。该项目的目标是了解Slo2.1通道激活的分子细节，并确定它们在缺血期间在心脏不同层和不同区域之间发展的电兴奋性梯度中的作用。这些电梯度与经历过心脏骤停的人的心脏除颤和复苏的结果高度相关。

DESCRIPTION (provided by applicant): Large conductance K+ channels activated by elevated levels of intracellular Na+ were first described in the heart in 1984. At that time it was postulated that the current (IKNa) conducted by these channels was most likely only activated during severe ischemia and has since been largely ignored. Six years ago, the channel that conducts IKNa was cloned and named Slo2.1. However, little is known regarding the structural basis of Slo2.1 channel function, including the molecular basis of the weak voltage dependence of channel activation, the location of the activation gate, or the mechanisms that couple intracellular Na+ binding to channel opening. Our preliminary findings indicate two novel features of Slo2.1. First, the usual voltage-sensor (S1-S4 segments) has no role in sensing transmembrane voltage. Second, the selectivity filter, not the S6 bundle crossing, may function as the activation gate. In Aim1 we will use site-directed mutagenesis, heterologous expression in Xenopus oocytes and voltage clamp techniques to substantiate these initial findings and definitively describe the molecular basis of Slo2.1 channel activation. Cardiac arrest is often associated with a combination of ventricular fibrillation (VF) and ischemia (VF/ischemia), both conditions promoting a rise in [Na+]i. A potential and yet unexplored role for IKNa during VF/ischemia is its contribution to depression and loss of excitability via an increase in the net outward current and extracellular K+ accumulation. During prolonged VF/ischemia, large gradients of activation rate and excitability develop between the enodcardium and the epicardium and between the right and the left ventricle (RV and LV), which are highly relevant to the outcomes of defibrillation and post-shock resuscitation. Yet the exact mechanism of these gradients remains a puzzle. Our preliminary results reveal regional differences in Slo 2.1 expression which may explain the electrophysiological gradients. There is also direct and indirect evidence supporting an important role of IKNa in outward K+ leak during ischemia. Unfortunately, knowledge of the role of IKNa in ischemia or VF/ischemia is very limited. In Aim 2 we will test the hypothesis that in the dog heart, the LV epicardium is the first region to exhibit inexcitability during VF/ischemia because myocytes in this region either accumulate cytoplasmic Na+ faster or express a greater density of IKNa channels compared to the LV endocardium or RV/septum. KNa channels are activated during ischemia, reperfusion and rapid heart rates when [Na+]i is transiently increased. In Aim 3, activation of KNa channels under these conditions will be examined in isolated canine ventricular myocytes. Finally, the relative contributions of IKNa and IKATP in these phenomena will also be investigated in the whole heart (Aim 2) and in isolated myocytes (Aim 3) of dogs. Together these studies will define 1) the molecular basis of KNa channel activation, 2) the role of these channels under normal and pathophysiological conditions and 3) the potential role of these channels as modulators of defibrillation and post-shock resuscitation. PUBLIC HEALTH RELEVANCE: Ion channels are membrane-bound proteins that selectively conduct specific ions in and out of cells. A plethora of potassium-selective ion channels are expressed in the human heart and are important components of the electrical activity that is responsible for the normal pump function of this organ. One type of potassium ion channel is called Slo2.1 and these channels are only activated if the intracellular concentration of sodium is elevated to abnormal levels as can occur during ischemia. Myocardial ischemia is both a frequent cause and a consequence of ventricular tachycardia and fibrillation, two potentially lethal forms of cardiac arrhythmia. The goals of this project are to understand the molecular details of Slo2.1 channel activation and define their role in the gradient of electrical excitability that develops between various layers and regions of the ventricle in the heart during ischemia. These electrical gradients are highly relevant to the outcomes of cardiac defibrillation and resuscitation of individuals who have experienced sudden cardiac arrest.