FUNCTION AND REGULATION OF NA/CA EXCHANGERS

Na/Ca交换是强有力的Ca转运体，其强烈影响 心脏收缩和电活动，以及分泌和 其他组织的收缩。我们的目标是更好地理解 心脏（NXC 1）Na/Ca交换功能和调节。耦合到此 目标是两个超越提案范围的进一步目标： 开发改进的方法来研究离子迁移，包括 电子中性转运蛋白;以及对磷脂酰肌醇的理解 控制表面膜离子转运的信号传导。1)运输 功能为了通过嵌合方法进行结构-功能研究， 鱿鱼NCX 1同源物的转运和门控特性改变 （NCX-SQ 1）、果蝇（Calx）和拟南芥。验证 离子传输的交替访问模型，我们将测试是否 突变为半胱氨酸或组氨酸的单个残基改变其 当离子被输送时，从内到外的可达性。（二） 交换器调节。交换器调制的分子基础 将分析磷脂酰肌醇-4 '-5'-二磷酸（PIP 2）， 交换器调节机制将根据初步探讨 指示在（a）类型2的水平上的潜在调节的数据 磷脂酰肌醇-4 '-激酶，（B）Gq调节的磷脂酶C，（c） 脂质磷酸酶，和（d）PIP 2亲和力的调节变化。第三章 细胞功能。将对假设进行检验， 频率和拉伸调节Na/Ca交换活性，从而 收缩性，通过PIP 2的变化。我们提出的研究有三个 重要的方法学组成部分：1）巨斑和全细胞 改进了Na/Ca交换调节的研究方法。（二） 光纤方法已经发展到监测离子传输活动 通过人为限制的细胞外 空间它们将通过测量Na/H交换活性进行验证， 它们将用于测试可能的交换化学计量变化 并验证拟南芥交换剂的不同离子选择性。 3）。已经开发出一种灵敏的非放射性方法来量化 磷脂，包括磷脂酰肌醇。总的来说， 因此，我们的建议具有广泛的生理意义， 与Ca在心肌缺血中的病理作用高度相关。

Na/Ca exchanges are powerful Ca transporters which strongly influence cardiac contractile and electrical activity, as well as secretion and contraction in other tissues. Our goal is an improved understanding of cardiac (NXC1) Na/Ca exchange function and regulation. Coupled to this goal are two further goals which transcend the scope of the proposal: the development of improved methods to study ion transport, including electroneutral transporters; and an understanding of phosphatidylinositol signaling in the control of surface membrane ion transport. 1) Transport function. To allow structure-function studies by a chimeric approach, the altered transport and gating properties of NCX1 homologues from squid (NCX-SQ1), drosophila (Calx), and arabidopsis will be analyzed. To verify an alternating access model of ion transport, we will test whether individual residues, mutated to cysteine or histidine change their accessibility from inside to outside when ions are transported. 2) Exchanger regulation. The molecular basis of exchanger modulation by phosphatidylinositol-4'-5'-biphosphate (PIP2) will be analyzed, and exchanger regulatory mechanisms will be explored in light of preliminary data indicating potential regulation at the levels of (a) type 2 phosphatidylinositol-4'-kinase, (b) Gq-regulated phospholipase C, (c) lipid phosphatases, and (d) regulatory changes of PIP2 affinity. 3) Cellular function. The hypotheses will be tested that changes of cardiac frequency and stretch modulate Na/Ca exchange activity, and thereby contractility, via changes of PIP2. Our proposed studies has three significant methodological components: 1) Giant-patch and whole-cell methods for studies of Na/Ca exchange regulatory have been improved. 2) Fiber optic methods have been developed to monitor ion transport activity via concentration changes in an artificially restricted extracellular space. They will be validated by measurements of Na/H exchange activity, and they will be used to test for possible exchange stoichiometry changes and to verify different ion selectivities of the Arabidopsis exchanger. 3). A sensitive, non-radioactive method has been developed to quantify phospholipids, including phosphatidylinositides, on TLC plates. In total, therefore, our proposal is of broad physiological significance and is highly relevant to pathological roles of Ca in cardiac ischemia.