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

巨型膜片中的 NA /CA 交换系统

• 批准号: 2228004
• 负责人: DONALD W HILGEMANN
• 金额: $ 20.64万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2228004
• 关键词: Xenopus; Xenopus oocyte; active sites; adenosine triphosphate; biological transport; calcium transporting ATPase; cytoskeleton; fluorescent dye /probe; guinea pigs; heart cell; lipid bilayer membrane; magnesium; mathematical model; membrane potentials; membrane transport proteins; phospholipids; sarcolemma; sodium potassium exchanging ATPase; voltage gated channel

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.

该提议的主要目的是了解机制和 心脏钠钙（NA/CA）的次级调节特性 交换过程。 计划实验以建立 从 交换周期的部分反应至交换器的水平 与肌膜膜的相互作用。 实验将 特别研究1）生物物理基础和动力学 运输周期中的电源反应，2） 次级调制（“门控”）反应及其在完整中的特性 肌细胞，3）克隆的选定突变体的功能特性 交换器，4）交换器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 易位酶。 这些领域的每个领域的拟议工作将利用 该实验室中开发了创新的实验技术，特别是 巨型切除的膜贴剂方法（15-40妈妈直径斑块; 5-18 pf; 1-10 Gomego密封）。 研究的研究 交换器将利用电压夹的速度（AP 5 MUS）和溶液 切换（AP。10ms），目前可以在巨大的补丁中实现。 到 研究磷脂组成深刻作用的机制 在交换电流上，巨大斑块的膜磷脂组成 在个人实验期间，将通过一种新方法修改 将磷脂通过疏水移液涂层转移到巨大的斑块中。 使用荧光“膜探针”的光学方法将用于 测试MGATP刺激心脏交换活性的假设 涉及建立磷脂酰丝氨酸（PS）不对称 氨基磷脂易位酶。 可以预料，这项工作将会 朝着对NA/CA交流的全面理解的进展 过程，在膜研究中采取更适当的方法论 运输，并了解生理上重要的 控制运输功能的控制机制。 知识 要获得的是对两者中心脏功能的理解至关重要的 生理和病理环境。