Regulation of the Cardiac Na/Ca Exchanger

钠/钙交换器将钙离子在质膜上的运动与钠离子在相反方向的运动耦合在一起。它是心肌细胞钙的主要外排机制，在控制心肌细胞钙含量和调节心肌收缩力方面起着重要作用。我们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，并阻断与调节性Ca激活相关的交换器的构象转变。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以一种专门针对交换剂调节特性的方式抑制交换活性。交换活性将在表达心脏Na/Ca交换的转染CHO细胞和新生大鼠心肌细胞中使用荧光胞浆钙指示剂测量。该项目有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)将开发一种新的交换剂调节动力学公式，具体地将称为“na依赖性失活”的过程与调节ca活化的变化联系起来。(3)当胞质钙升高或内部钙储存被填满时，神经酰胺抑制的发展速度将被测量，以检验神经酰胺抑制需要交换器形成缺钙构象的假设。其他的研究将验证在没有Ca活化的情况下神经酰胺不会抑制交换器的预测。(4)神经酰胺通过干扰脂筏和/或小泡等侧脂质亚域抑制交换活性的假设将得到验证。(5)本研究旨在验证肌动蛋白细胞骨架的药理稳定将交换器锁定在激活状态，降低其对胞质Ca调节或神经酰胺抑制的易感性。(6)神经酰胺抑制交换活性的生理意义将通过测量神经酰胺对(a)交换活性、(b)钙瞬态和(c)跳动的新生大鼠心肌细胞肌浆网中钙含量的影响来评估。其他研究将评估交换活性的抑制是否有助于TNFalpha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.