BIOCHEMICAL STUDIES ON SLOW CA2+ CHANNELS IN HEART CELLS

心脏细胞中动作电位的平台期是由于 Ca 2+离子通过电压依赖性通道流入。 这些通道 已经被广泛地研究了电生理学， 它们是时间和电压依赖性的，它们被正性肌力调节 激素（例如，去甲肾上腺素、组胺、血管紧张素II）和药物（例如， 维拉帕米、硝苯地平）。 关于这一过程的生物化学信息很少。 渠道或其监管，这种缺乏数据刺激了拟议的 工作 血管紧张素II（AII），一种正性肌力激素和Ca+2通道 agoinst，将被用作探针来研究Ca+2的激素机制 信道调制 高亲和力AII受体（Kd=0.59 nM）已被证实是 在悬浮培养的大鼠心肌细胞中发现， 将扩展到125 I-AII结合的表征， 培养的心肌细胞自发搏动。 这些功能反应 将通过测量动作电位的变化来识别AII细胞 在这些细胞中使用单电极记录方法。 生化 将通过检查AII的变化来探索肌细胞对AII的反应。 跨肌膜Ca+2通量、cAMP水平和蛋白质磷酸化。 这些实验的设计将使直接的相关性， 检查AII受体占用率、功能反应和 在相同条件下，完整培养的心肌细胞的生化反应 条件 Ca ~（2+）拮抗剂将被用作探针进行药理学研究 调节Ca+2通道活性。 带电的，不渗透的 将合成维拉帕米、地尔硫卓、硝苯地平和苄普地尔， 探索这些药物具有细胞内位点的可能性 行动上 这些化合物的相对效力时，应用内- 与细胞外的进行比较。 Ca+2的分子组成 膜和完整肌细胞中的降钙素受体将通过以下方法鉴定： 从羧基硝苯地平衍生物合成光亲和标记物 申请人最近提出的。 在进一步的研究中，羧基硝苯地平 将与琼脂糖凝胶珠偶联。 该支持将用于亲和性 用于鉴定高亲和力蛋白质的色谱程序 硝苯地平受体 长期目标是表征激素和 药理学调节心脏细胞中的慢Ca+2通道， 应该提供兴奋-收缩耦合过程的信息。 这项研究的成功完成将使人们了解 许多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.