Physiology and Biophysics of Cardiac Slo2.1 Channels

描述（由申请人提供）：1984年首次在心脏中描述了由细胞内Na+水平升高激活的大电导K+通道。当时，据推测，这些通道传导的电流（IKNa）很可能只在严重缺血期间被激活，此后在很大程度上被忽略了。六年前，传导IKNa的通道被克隆并命名为Slo2.1。然而，关于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、激活门的位置或将细胞内Na+结合与通道开放耦合的机制。我们的初步研究结果表明Slo2.1的两个新功能。首先，通常的电压传感器（S1-S4段）在感测跨膜电压中没有作用。第二，选择性过滤器，而不是S6束交叉，可以用作激活门。在目标1中，我们将使用定点诱变，异源表达在非洲爪蟾卵母细胞和电压钳技术，以证实这些初步的研究结果，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）的组合相关，这两种情况都会促进[Na+]i升高。IKNa在VF/缺血期间的一个潜在但尚未探索的作用是其通过增加净外向电流和细胞外K+积累而对抑郁和兴奋性丧失的贡献。在长时间VF/缺血期间，在内膜和心外膜之间以及在右心室和左心室（RV和LV）之间产生大的激活率和兴奋性梯度，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表达的区域差异，这可以解释电生理梯度。也有直接和间接的证据支持IKNa在缺血期间外向K+渗漏中的重要作用。不幸的是，IKNa在缺血或VF/缺血中的作用的知识非常有限。在目标2中，我们将检验以下假设：在犬心脏中，LV心外膜是VF/缺血期间表现出不兴奋性的第一个区域，因为与LV内膜或RV/隔膜相比，该区域中的肌细胞要么更快地积累细胞质Na+，要么表达更高密度的IKNa通道。 当[Na+]i短暂增加时，KNa通道在缺血、再灌注和快速心率期间被激活。在目标3中，将在分离的犬心室肌细胞中检查这些条件下KNa通道的激活。最后，IKNa和IKATP在这些现象中的相对贡献也将在犬的整个心脏（目的2）和分离的肌细胞（目的3）中进行研究。这些研究将共同定义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调节剂的潜在作用。 公共卫生相关性：离子通道是一种膜结合蛋白质，可选择性地将特定离子导入和导出细胞。过多的钾选择性离子通道在人类心脏中表达，并且是负责该器官的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，并且这些通道仅在细胞内钠浓度升高至异常水平时被激活，如在缺血期间可能发生的。心肌缺血是室性心动过速和室颤的常见原因和结果，室性心动过速和室颤是两种潜在的致命形式的心律失常。该项目的目标是了解Slo2.1通道激活的分子细节，并定义它们在缺血期间心脏心室各层和区域之间发展的电兴奋性梯度中的作用。这些电梯度与经历心脏骤停的个体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.