STRUCTURE OF ATP SENSITIVE POTASSIUM CHANNELS

描述(改编自研究人员的应用)：对ATP敏感的K+ 通道KATP可以由ATP结合的成员SUR1重组 盒式磁带超家族和KIR6.2，内向整流K+通道的成员 一家人。重建的通道是温和的内向整流器，其 电导受ATP/ADP、磺酰尿素通道的适当调节 阻滞剂和钾通道开放剂。初步数据显示，SUR1和 KIR6.2以1：1的化学计量比组装成大型复合体，假设 KATP通道。该应用程序的总体目标是测试 假设KATP通道具有四聚体结构， (SUR1/KIR6.2)4.具体目标是：1)建立SUR1和 KIR6.2结合形成异多聚体复合体。协会正在被 使用His标记的SUR1和KIR6.2亚基，通过利用特定的 结合SUR1的凝集素，通过125I-叠氮格列本脲和125I-叠氮格列本脲和 通过出现SUR1的复杂糖基化模式。2)至 测定络合物的化学计量比。这是通过以下方式完成的 沉降速度测量以估计分子大小(S) 并通过工程和表达活性通道形成融合 具有明确化学结构的蛋白质，例如SUR1-KIR6.2和 SUR1-(KIR6.2)2.3)，以确定活性KATP通道的化学计量比。 这是使用KIR6.2的突变体N160D完成的，该突变体赋予了强大的 对重组的KATP通道的整顿。野生型共表达 具有N 160D突变的KIR6.2提供了具有以下特性的异构体通道， 以及SUR1-N160D融合通道，将用于确定是否处于激活状态 通道是四聚体。4)开始绘制交互的领域 在SUR1和KIR6.2之间。这是由KIR6.2/KIR3.4工程部门完成的 嵌合体以确定关联需要KIR6.2的哪些部分以及 其用于形成活动通道。KIR3.4类似于6.2，但 不与SUR1关联或形成活动通道。这项工作将提供一种 三磷酸腺苷结合盒成员的离子通道调节模型 超级大家庭。

DESCRIPTION (Adapted from the investigator's application): ATP-sensitive K+ channels, KATP, can be reconstituted from SUR1, a member of the ATP-binding cassette superfamily and KIR6.2, a member of the inward rectifier K+ channel family. The reconstituted channel is a moderate inward rectifier whose conductance is appropriately modulated by ATP/ADP, by sulfonylurea channel blockers and potassium channel openers. Preliminary data suggest SUR1 and KIR6.2 assemble with a 1:1 stoichiometry into large complexes, presumably KATP channels. The overall objective of the application is to test the hypothesis that KATP channels have a tetrameric architecture, (SUR1/KIR6.2)4. The specific objectives are: 1) To establish that SUR1 and KIR6.2 associate to form a heteromultimeric complex. Association is being monitored using his-tagged SUR1 and KIR6.2 subunits, by utilizing specific lectins that bind SUR1, by cophotolabeling with 125I-azidoglibenclamide and by the appearance of a complex glycosylation pattern of SUR1. 2) To determine the stoichiometry of the complex. This is being done using sedimentation velocity measurements to estimate the molecular size(s) of the complexes and by engineering and expressing active channel forming fusion proteins with defined stoichiornetries, e.g., SUR1-KIR6.2 and SUR1-(KIR6.2)2. 3) To determine the stoichiometry of active KATP channels. This is being done using a mutant of KIR6.2, N 160D, that confers strong rectification on reconstituted KATP channels. Co-expression of wildtype KIR6.2 with the N 160D mutation gives heteromeric channels whose properties, along with SUR1-N160D fusion channels, will be used to determine if active channels are tetrameric. 4) To start to map the domains of interaction between SUR1 and KIR6.2. This is being done by engineering KIR6.2/KIR3.4 chimeras to determine which parts of KIR6.2 are needed for association and which for formation of active channels. KIR3.4 is similar to 6.2, but does not associate or form active channels with SUR1. This work will provide a model for ion channel regulation by members of the ATP-binding cassette superfamily.