Molecular Anatomy of Cardiac Ion Channels by Developing and Applying New Molecular Tools

心血管疾病是现代社会中致死率最高的严重健康问题。为了诊断患者，揭示其原因，并建立更好的治疗方法，有必要揭示与心脏兴奋和传播相关的分子的结构和功能。我们用化学、生物化学、免疫化学和电生理等多种方法研究了三种心脏通道（Ca^2+通道、Na^+通道和ATP敏感性K^+通道）。经过2年的工作，取得的成果总结如下。(1)用一种新合成的试剂进行光亲和标记，成功地确定了典型钙拮抗剂二氢吡啶在Ca^2+通道中的结合位点。所揭示的位点与骨骼肌对应物相同，但在位点中观察到氨基酸的几个取代。它们可能意味着两个通道之间的结合亲和力相差10倍。(2)通过对用新型格列本脲衍生物光标记的心脏标本进行生化纯化，分离出一种ATP敏感性K^+通道的潜在候选蛋白。免疫组织化学实验表明，该蛋白定位于膜表面的心脏小心室。(3)合成了用于光亲和标记和抗体生成的美西律类似物，作为心脏Na^+通道的典型I类抗心律失常药物的分子探针。

Cardiovascular diseases are serious problems in health ranking the highest lethal rate in modern society. In order to diagonize the patients, reveal their causes, and establish better therapeutic methods, it is essential to reveal structures and functions of molecules that are relevant to cardiac excitation and propagation. We investigated three cardiac channels (Ca^<2+>channel, Na^+channel, and ATP-sensitive K^+channel) by various methods including chemical, biochemical, immunochemical, and electrophysiological techniques. The results obtained after 2 years works are summarized as follows.(1) We are succeeded in identifying the binding sites of a typical calcium antagonist (dihydropyridine) in Ca^<2+>channel by photoaffinity labeling with a newly synthesized reagent. The sites revealed were identical to the skeletal muscle counterpart, but several substitution in amino acids was observed in the sites. They may imply the 10-fold difference of binding affinity between the two channels.(2) A potential candidate protein of ATP-sensitive K^+channel was isolated by biochemical purification of the cardiac preparations that were photolabeled with a new glibenclamide derivative. Immunohisto-chemical experiments showed that the protein was localized in the membrane surface of the ardiac venticules.(3) Mexiletine analogs for photoaffinity labeling and antibody generation were synthesized as molecular probes of a typical class I anti-arrhythmic agent for cardiac Na^+channels.