BIOCHEMICAL STUDIES ON SLOW CA2+ CHANNELS IN HEART CELLS

心肌细胞动作电位的平台期是由于 钙离子通过电压依赖通道的内流。这些渠道 已经被广泛地研究了电生理学和除了 它们依赖于时间和电压，受到变力作用的调制 激素(如去甲肾上腺素、组胺、血管紧张素II)和药物(如 维拉帕米、硝苯地平)。几乎没有什么生化信息 渠道或其监管，这种数据的缺乏阻碍了拟议的 工作。 正性肌力激素血管紧张素II与钙通道 Agoinst，将被用作研究Ca+2激素机制的探针 频道调制。高亲和力AII受体(Kd=0.59 nM) 在培养的大鼠心肌细胞中被鉴定，在悬浮液中，现在这些研究 将扩展到125I-AII结合到 自发地击打培养的心肌细胞。它们的功能反应 AII细胞将通过测量动作电位的变化来识别 在这些电池中使用单电极记录方法。生物化学 心肌细胞对AII的反应将通过检测 跨肌膜Ca+2流量、cAMP水平和蛋白质磷酸化。 这些实验将被设计成可以直接关联 检查AII受体占有率、功能反应和 原代培养的心肌细胞在相同温度下的生化反应 条件。 钙离子拮抗剂将作为探针用于药理研究 钙离子通道活性的调节。被指控的，非故意的衍生品 维拉帕米、地尔硫卓、硝苯地平和苯丙地尔将被合成到 探索这些药物在细胞内具有 行动。这些化合物在体内应用时的相对效力- 与细胞外的进行比较。钙离子的分子组成 膜和完整心肌细胞中的Anagoinst受体将通过 羧基硝苯地平衍生物合成光亲和标记 申请人最近准备的。在进一步的研究中，羧基硝苯地平 会被偶联到琼脂糖珠上。这种支持将在亲和力中使用 鉴定高亲和力蛋白质的层析程序 硝苯地平受体。 长期的目标是描述荷尔蒙和 心肌细胞慢钙通道的药理调节 应提供有关兴奋-收缩耦合过程的信息。 这项研究的成功完成将使人们对 一些心脏活性药物和激素的作用机制。

The plateau phase of the action potential in heart cells is due to an influx of Ca2+ ions through voltage dependent channels. These channels have been extensively studied electro-physiologically and in addition to their being time-and voltage-dependent, they are modulated by inotropic hormones (eg. norepinephrine, histamine, angiotensin II) and drugs (eg. verapamil, nifedipine). There is little biochemical information on the channel or its regulation and this lack of data has stimualted the proposed work. Angiotensin II (AII), a positive inotropic hormone and Ca+2 channel agoinst, will be used as a probe to study hormonal mechanisms of Ca+2 channel modulation. High affinity AII receptors (Kd=0.59 nM) have been identified in cultured rat myocytes, in suspension, and now these studies will be extended to the characterization of 125I-AII binding to spontaneously beating cultured myocytes. The functional responses of these cells to AII will be identified by measuring changes in action potentials in these cells using single-electrode recording methods. The biochemical responses of myocytes to AII will be explored by examining changes in transsarcolemmal Ca+2 fluxes, cAMP levels, and protein phosphorylation. These experiments will be designed so that direct correlations can be examined between AII receptor occupancy, functional responses, and biochemical responses of the intact cultured myocytes under identical conditions. The Ca+2 antagonists will be used as probes to study pharmacological regulation of Ca+2 channel activity. Charged, impermeant derivatives of verapamil, diltiazem, nifedipine, and bepridil will be synthesized to explore the possibility that these drugs have intracellular sites of action. The relative potencies of these compounds when applied intra- versus extracellularly will be compared. Molecular components of the Ca+2 antagoinst receptor in membranes and intact myocytes will be identified by synthesizing a photoaffinity label from carboxy nifedipine, a derivative recently prepared by the applicant. In further studies, carboxy nifedipine will be coupled to Sepharose beads. This support will be used in affinity chromatographic procedures to identify proteins of the high affinity nifedipine receptor. The long range goal is to characterize molecular mechanisms of hormonal and pharmacological regulation of slow Ca+2 channels in heart cells which should provide information on the excitation-contraction coupling process. The successful completion of this study will give insights into the mechanism of action of a number of cardioactive drugs and hormones.