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

巨型膜片中的 NA /CA 交换系统

• 批准号: 2228003
• 负责人: DONALD W HILGEMANN
• 金额: $ 22.32万
• 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/2228003
• 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.

该提案的主要目标是了解该机制并 心脏钠钙 (Na/Ca) 的次级调节特性 交换过程。 该实验计划是为了建立一个 从以下层面对交换器功能进行有凝聚力的综合描述 交换循环的部分反应直至交换器水平 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）详细功能 二次调制（“门控”）反应及其完整的特性 肌细胞，3）所选克隆突变体的功能特性 交换器，4）交换器与其脂质和细胞骨架的相互作用 环境，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。 每个领域的拟议工作将利用 该实验室开发的创新实验技术，特别是 巨型切除膜贴片法（15-40μm直径贴片；5-18pF； 1-10 戈麦戈密封件）。 部分生电反应的研究 交换器将利用电压钳的速度（ap. 5 mus）和解决方案 切换（大约10毫秒）目前可以在巨大的补丁中实现。 到 研究磷脂成分产生深远影响的机制 交换电流、巨斑块膜磷脂组成 将在个别实验中通过一种新方法进行修改 通过疏水性移液管涂层将磷脂转移到大斑块上。 采用荧光“膜探针”的光学方法将用于 检验 MgATP 刺激心脏交换活动的假设 涉及通过磷脂酰丝氨酸（PS）不对称性的建立 氨基磷脂转位酶。 预计这项工作将 对 Na/Ca 交换的全面理解取得进展 过程，寻求更充分的膜研究方法 运输，并了解生理上重要的 控制运输功能的调节机制。 知识点 获得的知识是了解心脏功能的基础 生理和病理环境。