FUNCTION AND REGULATION OF NA/CA EXCHANGERS

钠/钙交换是强大的钙转运体，它对 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(Nxc1)Na/Ca交换功能及其调节。再加上这个 目标是超越提案范围的另外两个目标： 研究离子传输的改进方法的发展，包括 电子中和转运体；对磷脂酰肌醇的理解 信号转导在控制表面膜离子转运中的作用。1)交通运输 功能。为了能够通过嵌合方法进行结构-功能研究， 鱿鱼NCX1同系物的运输和门控特性改变 (NCX-SQ1)、果蝇(Calx)和拟南芥将被分析。要验证 离子传输的交替访问模型，我们将测试 突变为半胱氨酸或组氨酸的单个残基改变其 当离子被传输时，从内到外的可访问性。2) 交易所监管。交换器调节的分子基础 将分析磷脂酰肌醇-4‘-5’-二磷酸(PIP2)，以及 将根据初步情况探讨交易所监管机制 数据表明在(A)类型2的水平上的潜在管制 磷脂酰肌醇-4‘-激酶，(B)Gq调节的磷脂酶C，(C) 脂质磷酸酶，以及(D)PIP2亲和力的调节变化。3) 细胞功能。这些假说将被检验为心脏的变化 频率和伸展调节Na/Ca交换活性，从而 收缩能力，通过改变PIP2。我们建议的研究有三个 重要的方法学组成部分：1)巨型补丁和全细胞 研究Na/Ca交换调节的方法已有所改进。2) 已经开发了光纤方法来监测离子传输活动 通过人工限制的胞外浓度的变化 太空。它们将通过测量Na/H交换活性来验证， 它们将被用来测试可能的交换化学计量学变化 并验证了拟南芥交换子的不同离子选择性。 3)。已经开发出一种灵敏的、非放射性的方法来量化 薄层板上的磷脂，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， 与钙在心肌缺血中的病理作用密切相关。

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.