Physiology and Biophysics of Cardiac Slo2.1 Channels

描述（由申请人提供）：1984年在心脏中首次描述了由细胞内Na+水平升高激活的大电导K+通道。当时的假设是，这些通道传导的电流（IKNa）很可能只在严重缺血时被激活，此后在很大程度上被忽视了。6年前，进行IKNa的频道被克隆并命名为Slo2.1。然而，关于Slo2.1通道功能的结构基础，包括通道激活弱电压依赖性的分子基础，激活门的位置，或细胞内Na+结合与通道打开的耦合机制，我们知之甚少。我们的初步发现表明了Slo2.1的两个新特征。首先，通常的电压传感器（S1-S4段）在感知跨膜电压方面没有作用。其次，选择性过滤器，而不是S6束交叉，可能起激活门的作用。在Aim1中，我们将使用位点定向诱变、非洲爪蟾卵母细胞中的异种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。心脏骤停通常与心室颤动（VF）和局部缺血（VF/ischemia）相结合，这两种情况都会促进[Na+]i升高。在VF/缺血过程中，IKNa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K+积累来促进抑郁和兴奋性丧失。在长时间的VF/缺血过程中，心内膜和心外膜之间、右心室和左心室之间（RV和LV）的激活率和兴奋性出现了较大的梯度，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，这可能解释了电生理梯度。也有直接和间接证据支持IKNa在缺血时向外K+泄漏中的重要作用。不幸的是，关于IKNa在缺血或VF/缺血中的作用的知识非常有限。在Aim 2中，我们将验证这样的假设：在狗心脏中，左室心外膜是第一个在VF/缺血期间表现不兴奋的区域，因为与左室心内膜或左室/隔膜相比，该区域的肌细胞要么更快地积累细胞质Na+，要么表达更大密度的IKNa通道。在缺血、再灌注和心率加快时，当[Na+]i瞬间升高时，KNa通道被激活。在Aim 3中，KNa通道在这些条件下的激活将在分离的犬心室肌细胞中进行检查。最后，IKNa和IKATP在这些现象中的相对贡献也将在狗的整个心脏（Aim 2）和分离的肌细胞（Aim 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，以及3)这些通道作为除颤和休克后复苏调节剂的潜在作用。

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.