REGULATION OF NA/K PUMP CURRENT IN THE HEART

描述(改编自申请者的描述)：Na/K泵建立 细胞在钠和钾中的跨膜梯度 兴奋性，Na-梯度中储存的能量由Na/Ca利用 交换以维持钙动态平衡。此外，泵产生一个向外的 直接影响心脏动作电位的电流。钠钾比的变化 泵活动可直接导致心律失常，或通过影响钠/钙 交换，导致钙超载和猝死。之前的研究由 申请者已经证明，豚鼠心室肌细胞共同表达 α1和α2亚型，具有异构体特异性的、依赖于钙的偶联 自动输入。这项提案涉及的一般问题是： 是这一复杂监管方案的生理目的，以及如何 不同的信号转导级联到每种异构体？ 初步数据提出了以下假设：β-肾上腺素能对 Na/K泵电流的大小是通过包含 α1-异构体；对电压依赖性的影响是通过直接 磷酸化和钙结合。α1-异构体主要被调节为 钙稳态：α2-异构体主要被调节以形成电 活动。目标1、2和3是研究信号转导的耦合 级联到特定的异构体。自主神经引起的细胞膜变化 电容将与泵电流的变化相关，同时干扰 与水泡交易有关。将测定异构体的磷化状态 使用P-32标记和磷酸特异性抗体。目标4、5和6是为了 探讨自主调节泵电流在钙稳态中的作用。 全细胞膜片钳技术将被用来表征Na/Ca 交换器与Na/K泵的钙依赖性。计算机模拟将会 有助于了解这两个运输系统与 细胞内钙离子。目标7和8是调查自主调节如何 泵的电流直接影响电活动。全细胞膜片钳 将被用来向心室动作电位注入电流，因此， 模拟自主神经调节的泵电流变化的直接影响。它 还将用于表征SA节点中泵电流的调节 起搏器细胞并确定泵电流的调节(特别是 α2亚型)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.