Regulation of the Cardiac Na/Ca Exchanger

Na/Ca交换器将Ca离子穿过质膜的运动与Na离子在相反方向上的运动耦合。 它是心肌细胞中主要的钙外流机制，在控制心肌细胞钙含量和调节心肌收缩力方面起重要作用。 对调节交换活动的细胞机制知之甚少。 在这个项目中，要研究的一般假设是，鞘脂是Na/Ca交换活性的生理调节剂，并阻止与交换器的调节Ca激活相关的构象转变。 这一假设是基于初步结果表明，短链神经酰胺类似物和鞘氨醇抑制交换活性的方式，是专门针对交换的监管性质。 在表达心脏Na/Ca交换蛋白的转染CHO细胞和新生大鼠心肌细胞中，使用荧光胞质Ca指示剂测量交换活性。本项目有6个具体目标：（1）完成外源性和内源性神经酰胺或鞘氨醇对转染细胞交换活性影响的初步表征。(2)一个新的动力学配方交换调节，具体联系的过程称为“钠依赖性失活”的变化，监管钙激活，将被开发。 (3)将测量当胞质Ca升高或当内部Ca储存被填充时神经酰胺抑制发展的速率，以检验神经酰胺抑制需要形成交换剂的Ca耗尽构象的假设。其他研究将测试神经酰胺在不存在Ca激活的情况下不抑制交换剂的预测。 (4)将检验神经酰胺通过扰动侧向脂质亚结构域（如脂筏和/或小窝）抑制交换活性的假设。 (5)在这一目标的研究将测试的想法，药理学稳定的肌动蛋白细胞骨架锁定在激活状态的交换器，降低其敏感性调节胞质钙或抑制神经酰胺。 (6)神经酰胺抑制交换活性的生理学意义将通过测量神经酰胺对搏动的新生大鼠心肌细胞中肌浆网中（a）交换活性、（B）Ca瞬变和（c）Ca量的影响来评估。 其他研究将评估交换活性的抑制是否有助于TNF α的心脏毒性作用。

The Na/Ca exchanger couples the movement of Ca ions across the plasma membrane to the movement of Na ions in the opposite direction. It is the principal Ca efflux mechanism in cardiac myocytes and plays an important role in controlling the Ca content of myocardial cells and in regulating cardiac contractility. Little is known about the cellular mechanisms that regulate exchange activity. In this project, the general hypothesis to be investigated is that sphingolipids are physiological regulators of Na/Ca exchange activity and block the conformational transition associated with regulatory Ca activation of the exchanger. This hypothesis is based on preliminary results demonstrating that short chain ceramide analogs and sphingosine inhibit exchange activity in a manner that is specifically targeted to the exchanger's regulatory properties. Exchange activity will be measured using fluorescent cytosolic Ca indicators in transfected CHO cells expressing the cardiac Na/Ca exchanger and in neonatal rat cardiac myocytes. The project has 6 specific aims: (1) Initial characterizations of the effects of exogenous and endogenous ceramide or sphingosine on exchange activity in the transfected cells will be completed. (2) A new kinetic formulation of exchanger regulation, which specifically links the process termed "Na-dependent inactivation" to changes in regulatory Ca-activation, will be developed. (3) The rate at which ceramide inhibition develops when cytosolic Ca is elevated or when internal Ca stores are filled will be measued in order to test the hypothesis that ceramide inhibition requires the formation of the Ca-depleted conformation of the exchanger. Other studies will test the prediction that ceramide does not inhibit the exchanger in the absence of Ca activation. (4) The hypothesis that ceramide inhibits exchange activity by perturbing lateral lipid subdomains such as lipid rafts and/or caveolae will be tested. (5) The studies in this aim will test the idea that pharmacological stabilization of the actin cytoskeleton locks the exchanger in an activated state, reducing its susceptibility to regulation by cytosolic Ca or inhibition by ceramide. (6) The physiological significance of ceramide inhibition of exchange activity will be assessed by measuring the influence of ceramide on (a) exchange activity, (b) the Ca-transient and (c) the amount of Ca in the sarcoplasmic reticulum in beating neonatal rat cardiac myocytes. Other studies will assess whether inhibition of exchange activity contributes to the cardiotoxic effects of TNFalpha.