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

背景：电压门控钾通道Kv1.5在维持膜电位中起关键作用，心脏Kv1.5作为各种抗心律失常药物的靶点具有特殊的临床意义。虽然蛋白质降解是调节通道蛋白功能的主要机制之一，但通过脉冲追踪分析、免疫荧光和膜片钳技术对转染的COS细胞和大鼠心房肌细胞中Kv1.5蛋白的降解机制知之甚少。表达的Kv1.5半衰期较短，为6.7h。蛋白酶体抑制剂MG132，而不是溶酶体抑制剂氯喹，显著延长了半衰期，增加了总蛋白和泛素化蛋白的水平。Kv1.5主要分布于内质网和高尔基体。MG132增加了这些区室中Kv1.5蛋白的水平，导致通过细胞表面Kv1.5的Ik_<ur>电流显著升高。brefeldine A和秋水仙碱均能消除MG132对Ik…More _<ur>电流的影响。与MG132一样，Na^+通道阻滞剂匹西卡因和利多卡因延长了Kv1.5的半衰期，增加了蛋白水平和IK_<ur>电流。K^+通道阻滞剂E-4031和Ca^<2+>通道阻滞剂维拉帕米都没有这些作用。同样，Na^+通道阻滞剂和MG132均可提高原代培养大鼠心房肌细胞的Kv1.5水平。这些Na^+通道阻滞剂在体外抑制20S蛋白酶体活性，并稳定了COS细胞中IkB2蛋白的表达。结论：Kv1.5被蛋白酶体泛素化并降解。Na^+通道阻滞剂匹西卡因和利多卡因通过模拟MG132的作用，稳定Kv1.5并增加Ik_<ur>电流，提示这些药物具有抑制K^+通道蛋白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药理活性，表明Na^+通道阻滞剂的作用并非仅针对Kv1.5，而可能扩展到注定要蛋白酶体降解的其他蛋白质。少

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