REGULATION OF NA/K PUMP CURRENT IN THE HEART

描述（改编自申请人的描述）：Na/K泵建立 细胞的跨膜Na和K梯度。 这些是必不可少的， 兴奋性，和能量存储在钠梯度是由钠/钙使用 交换以维持Ca稳态。 此外，泵产生向外的 直接影响心脏动作电位的电流。 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，导致钙超载和猝死。 以前的研究， 申请人已经表明豚鼠心室肌细胞共表达 α 1-和α 2-亚型，具有亚型特异性、Ca依赖性偶联 到自主输入。 本建议中涉及的一般性问题是： 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异构体？ 初步数据提出了以下假设：β-肾上腺素能对 Na/K泵电流的量是通过含有 α 1-亚型;对电压依赖性的影响是通过直接 磷酸化和Ca结合。 alpha 1-同种型主要被调节为 钙稳态：α 2-亚型的调节主要是为了形成电 活动 目的1、2和3是研究信号转导的耦合 级联产生特定的亚型。 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 与囊泡运输有关 将测定亚型的磷酸化状态 使用P-32标记和磷酸特异性抗体。目标4、5和6是： 研究泵电流的自主调节在钙稳态中的作用。 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， 胞内钙 目的7和8是研究如何自主调节 泵电流直接影响电活动。 全细胞膜片钳 将被用于将电流注入心室动作电位，因此， 模拟泵电流中自主调节变化的直接效应。 它 也将用于表征SA节点中泵电流的调节 起搏细胞，并确定是否调节泵电流（特别是 α 2同种型）影响起搏。

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.