Phospholemman and Na-Pump Function in Cardiac Myocytes

描述(由申请人提供)：Na/K-ATPase(NKA)是主要的Na排出途径，因此在[Na]i调节中是必不可少的。在心脏，[Na]i通过Na/Ca交换(NCX)关键地调节[Ca]i和收缩能力，这使得了解[Na]i的调节极其重要。Pylolemman(PLM或FXYD1)是FXYD家族中的一种跨膜蛋白，已知与NKA相关并调节NKA。PLM是心脏中唯一含量丰富的FXYD蛋白，是蛋白激酶A(PKA)和蛋白激酶C(PKC)磷酸化的主要底物。在最初的获奖期间，我们发现PLM调节NKA的方式类似于磷蛋白(PLB)调节SR Ca-ATPase(SERCA)的方式。也就是说，PLM通过降低NKA的[Na]i亲和力来抑制NKA，而PLM的磷酸化则解除了这种抑制。我们还发现，PLM的磷酸化和随之而来的NKA刺激是交感战斗或逃跑反应的组成部分(缓和[Na]i和细胞内钙负荷的上升，并限制钙超载诱导的心律失常)。我们发现PLM与NKA1PLM磷酸化(通过免疫沉淀)物理上相关，但在荧光共振能量转移(FRET)测量中，PLM磷酸化改变了PLM-NKA相互作用，PLM形成同源低聚物。所有这些都类似于SERCA-PLB。尽管PLM-NKA具有生理意义，但其相互作用机制尚不清楚。因此，这项更新提案的总体目标是从机制上破译PLM和NKA如何在功能上相互作用。在这里，我们将结合FRET，[Na]i和[Ca]i测量，膜片钳和分子生物学技术在心肌细胞和HEK 293细胞中。目的1研究NKA-PLM(及PLM-PLM复合体)的结构与功能。这一点很重要，有几个原因。首先，最近与FXYD相关的NKA晶体结构表明，NKA和FXYD上的一些位置在相互作用距离内(至少在晶体中的特定稳定构象中)，但这需要在活细胞膜中进行测试。这将在AIM 1中通过原位FRET测量、细胞内Co-IP和NKA活性测量进行测试。其次，尚不清楚PLM-PLM同源低聚物如何相互作用(例如，它们是否像PLB一样形成具有结构基础的稳定多聚体)，以及是否存在与PLM-NKA二聚体平衡的PLM多聚体发挥功能调节(如PLB-SERCA)。这也将在目标1中进行评估。目标2更狭隘地关注PLB(至少是与人类疾病相关的PLB人类突变体)可能被误导到肌膜并调节NKA的有趣的可能性。我们将测试WT PLB是否也可以与NKA活性相互作用和调节，如果它在肌膜上(反之，如果PLM在ER/SR中表达，是否可以与SERCA功能相互作用)。Aim 3研究了一些问题，这些问题来自我们新颖而令人惊讶的观察，即哇巴因消除了NKA和PLM或NKA之间的烦恼。这将提供关于NKA的二聚化(异源或同源)如何与其功能活性相关的重要信息，以及NKA在激酶信号级联中的作用。

DESCRIPTION (provided by applicant): Na/K-ATPase (NKA) is the main Na extrusion pathway and therefore is essential in [Na]i regulation. In the heart, [Na]i critically modulates [Ca]i and contractility via Na/Ca exchange (NCX), which makes understanding [Na]i regulation extremely important. Phospholemman (PLM or FXYD1) is a transmembrane protein of the FXYD family of proteins that are known to associate with and modulate NKA. PLM is the only FXYD protein abundant in the heart, where it is a major substrate for phosphorylation by protein kinase A (PKA) and C (PKC). During the initial award period, we showed that PLM regulates NKA similar to the way phospholamban (PLB) regulates SR Ca-ATPase (SERCA). That is, PLM inhibits NKA by reducing its [Na]i affinity, and PLM phosphorylation relieves this inhibition. We also showed that PLM phosphorylation and the consequent NKA stimulation is integral to the sympathetic fight or flight response (tempering the rise in [Na]i and cellular Ca load, and limiting Ca overload-induced arrhythmias). We found that PLM physically associates with NKA 1PLM phosphorylation (via immunoprecipitation), but in fluorescence resonance energy transfer (FRET) measurements, that PLM phosphorylation alters the PLM-NKA interaction and that PLM forms homo- oligomers. All of this resembles SERCA-PLB. Despite its physiological significance, the mechanism of PLM- NKA interaction is poorly understood. Thus, the overall goal of this renewal proposal is to decipher mechanistically how PLM and NKA interact functionally. Here we will combine FRET, [Na]i and [Ca]i measurements, patch-clamp and molecular biology techniques in cardiac myocytes and HEK 293 cells. Aim 1 focuses on the structure-function of the NKA-PLM (and PLM-PLM complex). This is important for several reasons. First, the recent NKA crystal structures with associated FXYDs suggest some sites on NKA and FXYDs that are within interaction distance (at least in the particular stable conformation in the crystals), but this requires testing in live cell membranes. That will be tested in Aim 1 by in situ FRET measurements, Co-IP and NKA activity measurements in cells. Second, it is unknown how PLM-PLM homo-oligomers interact (e.g. whether they form stable multimers with a structural basis like PLB) and whether there is a pool of PLM multimers in equilibrium with PLM-NKA dimers that exerts functional regulation (as for PLB-SERCA). That will be assessed in Aim 1 as well. Aim 2 focuses more narrowly on the intriguing possibility that PLB (at least PLB human mutants that are relevant to human disease) can be misdirected to the sarcolemma and regulate NKA. We will test whether WT PLB can also interact with and modulate NKA activity if it is in the sarcolemma (and conversely if PLM can interact functionally with SERCA if it is expressed in the ER/SR). Aim 3 examines questions that are inspired from our novel and surprising observation that ouabain abolishes FRET between NKA and either PLM or NKA. This will provide important information about how dimerization (hetero or homo) of NKA relates to its functional activity, and possibly also the role of NKA in kinase signaling cascades.