Investigation of pathophysiological properties of ion channels on cardiac sarcoplasmic reticulum.

(1)兰尼碱受体Ca^2+释放通道调控机制的研究为研究心肌肌浆网（SR）ryanodine受体Ca^2+释放通道的调控机制，我们从猪心肌中分离纯化了SR膜。这些程序是在威斯康星州大学科罗纳多博士的实验室完成的。将Ryanodine受体掺入人工脂质双层膜中，用电压钳技术记录通道活动。川野博士和平冈博士访问了科罗纳多博士的实验室，研究和讨论了从猪和牛心脏中纯化兰尼碱受体的过程。利用这些制剂，我们可以记录Ryanodine受体的通道活动。在本研究中，我们研究了Mg^<2+>对该通道的影响，发现Mg^<2+>以不同的机制阻断通道开放。也就是说，（1）Mg^<2+>通过与Ca^<2 +>在通道的Ca^<2 +>结合激活位点竞争而减少通道开放，（2）Mg^<2 +>通过与Ca^<2 +>结合激活位点竞争而减少通道开放。 ...更多信息 从而揭示了Mg^<2 +>对SR释放Ca^<2 +>的调控机制。(2)猪心肌肌浆网氯离子通道的阴离子选择性研究。我们曾报道心肌肌浆网上存在Cl-通道，该通道由环腺苷酸依赖性磷酸化激活。在这项研究中，我们研究了该通道的离子选择性，并比较该通道的孔特性的其他Cl-通道。由Goldman-Hodgikin-Katz方程计算的渗透率为Br->Cl->I->NO_3->F-。这些结果与囊性纤维化跨膜调节因子（CFTR）和肌膜上的心脏Cl-通道的结果相当。该Cl-通道的生物物理性质与CFTR的生物物理性质非常相似，表明孔具有对阴离子的中等高亲和力位点。Coronado博士发现，氯离子可诱导肌浆网Ca^2+释放。因此，我们在SR和Cl-通道上对这些通道之间的关系进行了很好的讨论。由于与Coronado博士的合作，我们可以显示肌膜通道和SR通道之间的新信号传导途径。少

(1) Investigation of regulatory mechanism of ryanodine receptor Ca^<2+> release channel. To investigate the regulatory mechanisms of ryanodine receptor Ca^<2+> release channel in cardiac sarcoplasmic reticulum (SR), we purified the SR membrane from pig hearts. These procedures were done in Dr.Coronado's lab at University of Wisconsin. The ryanodine receptors were incorporated into the artificial lipid bilayr and the channel activities were recorded by voltage clamp technique. Dr.Kawano and Dr.Hiraoka visited Dr.Coronado's lab to study and to discuss the procedure of purification of ryanodine receptors from pigs and bovine hearts. By using these preparations, we could record channel activities of ryanodine receptor. In this study we investigated Mg^<2+> effects on this channel and found that Mg^<2+> blocked the channel openings with diverse mechanisms. Namely, (1) Mg^<2+> reduced channel openings by competing with Ca^<2+> at the Ca^<2+> binding activating site of the channel and (2) by … More reducing the channel conductance.Thus, we showed the detail regulatory mechanisms of Ca^<2+> release from SR by Mg^<2+>.(2) Investigation of anion selectivity of a chloride channel in the porcine cardiac sarcoplasmic reticulum. We have reported that Cl-channel are present on cardiac sarcoplasmic reticulum which is activated by cyclic AMP dependent phosphorylation. In this study we examined the ion selectivity of this channel and compare the pore properties of this channel to those of other Cl-channels. Permeability ration calculated by Goldman-Hodgikin-Katz equation revealed the anion permeability sequence as Br->Cl->I->NO_3->F-. Those results were comparable to those of cystic fibrosis transmembrane regulator (CFTR) and the cardiac Cl-channel on the sarcolemma. The biophysical properties of this Cl-channel are very similar to those of the CFTR,suggesting the pore with a moderately high affinity site for anions. Therefor we speculate that cardiac SR Cl-channel may belong to the same family of Cl-channel as CFTR.Dr.Coronado found chloride-induced Ca^<2+> release from sarcoplasmic reticulum. Therefore, we had good discussions about the relationships between these channels on SR and Cl-channel. We could show the new signal tansduction pathway between channels on sarcolemma and those on SR,because of collaboration with Dr.Coronado. Less