NA+/CA++ EXCHANGE SYSTEM IN GIANT MEMBRANE PATCHES

本建议的主要目的是了解该机制， 心脏钠钙（Na/Ca）的次级调节特性 交换过程。 计划进行实验，以便建立一个 从交换机功能的层次， 交换循环的部分反应直到交换器的水平 与肌膜的相互作用。 实验将 具体研究1）生物物理基础和动力学 运输循环中的生电反应，2） 二次调制（“门控”）反应及其在完整 肌细胞，3）克隆的选择的突变体的功能特性 交换剂，4）交换剂与其脂质和细胞骨架的相互作用 环境，和5）细胞质MgATP刺激的机制 交换活性，特别是氨基磷脂的可能作用 转位酶 这些领域的拟议工作将利用 本实验室开发的创新实验技术，特别是 巨大切除膜贴片法（15-40 μ m直径贴片; 5-18 pF; 1-10 Gomego密封）。 部分产电反应的研究 交换器将利用电压钳位（AP. 5 mus）和溶液 切换（AP. 10 ms），这目前可以在巨大的补丁中实现。 到 研究磷脂成分的深刻影响的机制 对巨斑的交换电流、膜磷脂组成的影响 将在单独的实验中通过一种新的方法进行修改， 通过疏水移液管涂层将磷脂转移到巨大的斑块上。 采用荧光“膜探针”的光学方法将用于 验证MgATP刺激心脏交换活动的假设 涉及磷脂酰丝氨酸（PS）不对称性的建立， 氨基磷脂转位酶 可以预计，这项工作将 对Na/Ca交换的全面理解的进展 过程，朝着更充分的方法学在膜的研究 运输，以及对生理学重要性的理解 在控制运输功能的调节机制。 知识 是了解心脏功能的基础， 生理和病理环境。

The principle objective of this proposal is to understand the mechanism and secondary modulatory properties of the cardiac sodium-calcium (Na/Ca) exchange process. The experimentation is planned so as to establish a cohesive, integrated picture of exchanger function from the level of partial reactions of the exchange cycle up to the level of exchanger interactions with the sarcolemmal membrane. The experimentation will specifically investigate 1) the biophysical basis and kinetics of electrogenic reactions in the transport cycle, 2) the detailed function of secondary modulation ('gating') reactions and their properties in intact myocytes, 3) the functional properties of selected mutants of the cloned exchanger, 4) interactions of the exchanger with its lipid and cytoskeletal environment, and 5) the mechanism by which cytoplasmic MgATP stimulates exchange activity, in particular the possible role of aminophospholipid translocase. The proposed work in each of these areas will exploit innovative experimental techniques developed in this lab, in particular the giant excised membrane patch method (15-40 mum diameter patches; 5-18 pF; 1-10 Gomego seals). Studies of the partial electrogenic reactions of the exchanger will exploit the speeds of voltage clamp (ap. 5 mus) and solution switching (ap. 10 ms) which can presently be achieved in giant patches. To investigate the mechanisms for profound effects of phospholipid composition on exchange current, membrane phospholipid composition of giant patches will be modified during individual experiments by a novel method to transfer phospholipids to giant patches via the hydrophobic pipette coat. Optical methods employing fluorescent 'membrane probes' will be used to test the hypothesis that stimulation of cardiac exchange activity by MgATP involves the establishment of phosphatidylserine (PS) asymmetry by an aminophospholipid translocase. It may be expected that this work will progress toward a comprehensive understanding of the Na/Ca exchange process, toward more adequate methodologies in the study of membrane transport, and toward an understanding of physiologically important regulatory mechanisms in the control of transport function. The knowledge to be gained is fundamental to an understanding of cardiac function in both physiological and pathological settings.