Mechanisms on the degradation of ion channel proteins and their modification by antiarrythmic agents

离子通道蛋白的降解机制及其抗心律失常药物的修饰

• 批准号: 13670713
• 负责人: HISATOME Ichiro
• 金额: $ 1.98万
• 依托单位: Department of Cardiovascular Medicine, Tottori University Faculty of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670713/
• 关键词: ion channel; ubiquitin; proteasome; degradation; Na^+channel blocker; 再生; Kv1.5; 細胞内輸送

Background: The voltage gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential and cardiac Kv1.5 is of particular clinical importance as a target of various antiarrhythmic drugs. While protein degradation is one of the major mechanisms to regulate channel protein functions, little is known on the degradation mechanism of Kv1.5 proteins was estimated by pulse chase an alysis, immnofluorescence and patch clamp techniques in transfected COS cells and rat atrial myocytes. Expressed Kv1.5 had a short half-life time of 6.7h. Aproteasome inhibitor MG132, but not a lysosome inhibitor chloroquine, significantly prolonged the half-life time and increased the levels of both total proteins and ubiquitinated proteins. Kv1.5 was mainly localized in the endoplasmic reticulum and Golgi apparatus. MG132 increased the levels of Kv1.5 proteins in these compartments, causing a significant in Ik_<ur>currents through the cell-surface Kv1.5. The effect of MG132 on Ik … More _<ur>currents was abolished both by brefeldine A and colchicine. Like MG132, Na^+channel blockers pilsicainide and lidocaine prolonged the half-life time of Kv1.5 and increased both protein levels and IK_<ur>currents. Neither a K^+channel blocker E-4031 nora Ca^<2+>channel blocker verapamil exerted these effects. Similarly, both Na^+channel blockers and MG132 increased the level of Kv1.5 in primary-cultured rat atrial myocytes. These Na^+ channel blockers inhibited the 20S proteasome activity in vitro and also stabilized the IkB2 protein expressed in COS cells. Conclusion: Kv1.5 is ubiquitinated and degraded by the proteasome. Na^+ channel blockers pilsicainide and lidocaine, by mimicking the action of MG132, could stabilize Kv1.5 and increase Ik_<ur>currents, suggesting a novel pharmacological action of these agents to inhibitproteasomal degradation of K^+channel proteins. These drugs possess a novel pharmacological activity to inhibit the proteasome, suggesting effect of the Na^+channel blockers is not specific to Kv1.5 but may extend to other proteins destined to proteasomal degradation. Less

背景：电压门控性钾通道Kv1.5在膜电位的维持中起着关键作用，心脏Kv1.5作为多种抗心律失常药物的靶点具有特殊的临床意义。蛋白质降解是调节通道蛋白功能的主要机制之一，但通过脉冲追踪法、免疫荧光法和膜片钳技术研究Kv1.5蛋白在COS细胞和大鼠心房肌细胞中的降解机制知之甚少。表达的Kv1.5的半衰期较短，为6.7h。非蛋白酶体抑制剂MG132但不是溶酶体抑制剂氯喹，可显著延长半衰期，并增加总蛋白和泛素化蛋白水平。Kv1.5主要定位于内质网和高尔基体。MG132增加了这些隔室中Kv1.5蛋白的水平，导致通过细胞表面Kv1.5的Ik电流显著增加。MG132对Ik…的影响灯盏花素A和秋水仙碱均可消除更多的电流。与MG132一样，钠离子通道阻滞剂匹西卡胺和利多卡因延长了Kv1.5的半衰期，增加了Kv1.5的蛋白水平和Ik_l；Ur&gt；电流。K~(+)通道阻断剂E-4031和钙通道阻滞剂维拉帕米均不能发挥上述作用。同样，Na~+通道阻滞剂和MG132均可增加原代培养的大鼠心房肌细胞Kv1.5的水平。这些钠离子通道阻滞剂在体外抑制20S蛋白酶体的活性，并稳定COS细胞中表达的IkB2蛋白。结论：Kv1.5被蛋白酶体泛素化和降解。Na~+通道阻断剂Pilsicainide和利多卡因可模拟MG132的作用，稳定Kv1.5，增加Ik_lt；ur&gt；电流，提示这两种药物具有抑制K~+通道蛋白酶体降解的新药理作用。这些药物具有抑制蛋白酶体的新的药理活性，表明Na+通道阻滞剂的作用不是Kv1.5所特有的，而可能延伸到其他蛋白酶体的降解。较少

项目成果

Gias U.Ahmmed: "Analysis of Moricizine Block of Sodium Current in Isolated Guinea-Pig Atrial Myocytes : Atrio-Ventricular Difference of Moricizine Block"General Pharmacology. (in press).

Gias U.Ahmmed：“莫里西嗪阻滞钠电流在离体豚鼠心房肌细胞中的分析：莫里西嗪阻滞的心房-心室差异”一般药理学。

加藤克: "プロテアソーム阻害薬は細胞内輸送を修飾することでKv1.5チャネルを増加させる"米子医学雑誌. 53. 18-29 (2002)

Katsu Kato：“蛋白酶体抑制剂通过改变细胞内运输来增加 Kv1.5 通道”米子医学杂志 53. 18-29 (2002)。

Mariko Tsuboi: "MITOCHONDRIAL DNA DELETION ASSOCIATED WITH THE REDUCTION OF ADENINE NUCLEOTIDES IN HUMAN ATRIUM AND ATRIAL FIBRILLATION"European Journal of Clinical Investigation. 31. 1-9 (2001)

Mariko Tsuboi：“与人心房腺嘌呤核苷酸减少和心房颤动相关的线粒体 DNA 缺失”欧洲临床研究杂志。

Okamura Y.: "Abnormally High Expression of Proteasome Activator-gamma in Thyroid Neoplasm"J Clin Endocrinol Metab. Mar;88(3). 1374-1383 (2003)

Okamura Y.：“甲状腺肿瘤中蛋白酶体激活剂-γ 的异常高表达”J Clin Endocrinol Metab。

Toru Yatsuhashi: "L-Cysteine Prevents Oxidation-Induced Block of Cardiac Na^+ Channel via Interaction with Heart-Specific Cysteinyl Residents in the P-loop Region"Circulation Journal. 66(9). 372-376 (2003)

Toru Yatsuhashi：“L-半胱氨酸通过与 P 环区域中心脏特异性半胱氨酰居民的相互作用来防止氧化诱导的心脏 Na^ 通道阻塞”循环杂志。