REGULATION OF NA/K PUMP CURRENT IN THE HEART

心脏 NA/K 泵电流的调节

• 批准号: 6537165
• 负责人: Richard T Mathias
• 金额: $ 33.86万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537165
• 关键词: autonomic nervous system; biological signal transduction; calcium flux; cardiac myocytes; enzyme mechanism; guinea pigs; heart electrical activity; heart innervation; isozymes; laboratory rabbit; membrane potentials; neuroregulation; phosphorylation; protein kinase C; protein structure function; sodium potassium exchanging ATPase; tissue /cell culture; voltage /patch clamp

DESCRIPTION (adapted from applicant's description): The Na/K pumps establish the cell's transmembrane gradients in Na and K. These are essential for excitability, and the energy stored in the Na-gradient is used by Na/Ca exchange to maintain Ca homeostasis. Moreover, the pump generates an outward current that directly affects the cardiac action potential. Changes in Na/K pump activity can lead directly to arrhythmias, or through effects on Na/Ca exchange, lead to Ca overload and sudden death. Previous studies by the applicant have shown that guinea pig ventricular myocytes co-express the alpha1-and alpha2-isoforms, which have isoform specific, Ca-dependent coupling to autonomic input. The general questions addressed in this proposal are: What is the physiological purpose of this elaborate regulatory scheme, and how are the different signal transduction cascades coupled to each isoform? Preliminary data suggest the following hypotheses: Beta-adrenergic effects on the amount of Na/K pump current are through cycling of vesicles containing the alpha1-isoform; effects on voltage dependence are through direct phosphorylation and Ca-binding. The alpha1-isoform is regulated primarily for Ca homeostasis: the alpha2-isoform is regulated primarily to shape electrical activity. Aims 1, 2 & 3 are to investigate the coupling of signal transduction cascades to specific isoforms. Autonomic induced changes in membrane capacitance will be correlated with changes in pump current, while interfering with vesicle trafficking. The phospho-state of the isoforms will be determined using P-32 labeling and phospho-specific antibodies. Aims 4, 5 & 6 are to investigate the role of autonomic regulation of pump current in Ca homeostasis. The whole cell patch clamp technique will be used to characterize the Na/Ca exchanger and the Ca-dependence of the Na/K pump. Computer simulations will help to understand the interactions of these two transport systems with intracellular Ca. Aims 7 & 8 are to investigate how autonomic regulation of pump current directly affects electrical activity. The whole cell patch clamp will be used to inject current into ventricular action potentials, thus, mimicking the direct effects of autonomic mediated changes in pump current. It will also be used to characterize regulation of pump current in SA node pacemaker cells and determine if regulation of pump current (particularly the alpha2 isoform) affects pacemaking.

描述（改编自申请人的描述）：Na/K 泵建立 细胞的 Na 和 K 跨膜梯度。这些对于 兴奋性，储存在 Na 梯度中的能量被 Na/Ca 使用 交换以维持Ca稳态。 此外，泵还产生向外的 电流直接影响心脏动作电位。 Na/K 的变化 泵活动可直接导致心律失常，或通过影响 Na/Ca 交换，导致 Ca 超载和猝死。 之前的研究 申请人已经证明豚鼠心室肌​​细胞共同表达 α1-和 α2-异构体，具有异构体特异性、Ca 依赖性偶联 到自主输入。 该提案解决的一般问题是： 是这个精心设计的调节方案的生理目的，以及如何 不同的信号转导级联耦合到每个亚型？ 初步数据表明以下假设： β-肾上腺素能作用 Na/K 泵电流的量是通过含有 α1-异构体；对电压依赖性的影响是通过直接 磷酸化和 Ca 结合。 α1-亚型主要受调节 Ca 稳态：α2-亚型主要受调节以形成电 活动。 目标 1、2 和 3 是研究信号转导的耦合 级联成特定亚型。 自主神经引起的膜变化 电容将与泵电流的变化相关，同时干扰 与囊泡贩运。 异构体的磷酸化状态将被确定 使用 P-32 标记和磷酸化特异性抗体。目标 4、5 和 6 是 研究泵电流自主调节在 Ca 稳态中的作用。 全细胞膜片钳技术将用于表征 Na/Ca 交换器和 Na/K 泵的 Ca 依赖性。 计算机模拟将 有助于理解这两个运输系统的相互作用 细胞内Ca。 目标 7 和 8 是研究自主调节如何 泵电流直接影响电活动。 全细胞膜片钳 将用于将电流注入心室动作电位，因此， 模仿自主调节泵电流变化的直接影响。 它 还将用于表征 SA 节点中泵浦电流的调节 起搏器细胞并确定是否调节泵电流（特别是 α2亚型）影响起搏。