STRUCTURE OF ATP SENSITIVE POTASSIUM CHANNELS

描述（改编自研究者申请）：ATP敏感性K+ 通道，KATP，可以从SUR 1重建，SUR 1是ATP结合通道的成员。 cassette超家族和内向整流钾通道KIR6.2 家人 重建的通道是一个温和的内向整流器， ATP/ADP、磺酰脲通道、 阻断剂和钾通道开放剂。 初步数据表明，SUR 1和 KIR6.2以1：1的化学计量组装成大的复合物，据推测 KATP通道。 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） 具体目标是：1）确定SUR 1和 KIR6.2缔合以形成异多聚体复合物。 协会正在 使用His标记的SUR 1和KIR6.2亚基进行监测， 通过与125 I-叠氮格列本脲共光标记结合SUR 1的凝集素， 通过SUR 1的复杂糖基化模式的出现。 2)到 确定络合物的化学计量。 这是通过使用 沉降速度测量，以估计 复合物，并通过工程化和表达活性通道形成融合 具有确定化学计量的蛋白质，例如，SUR 1-KIR6.2和 SUR1-（KIR6.2）2. 3)确定活性KATP通道的化学计量。 这是使用KIR6.2的突变体N 160 D来完成的，其赋予强的 重组KATP通道的整流。 野生型共表达 具有N160 D突变的KIR6.2产生异聚体通道， 沿着SUR 1-N160 D融合通道，将用于确定是否激活 通道是四聚体。 4)开始绘制互动的领域 在SUR 1和KIR6.2之间。 这是由工程KIR6.2/KIR3.4完成的 嵌合体，以确定KIR6.2的哪些部分是关联所需的， 其用于形成活性通道。 KIR3.4类似于6.2，但 不与SUR 1关联或形成活动通道。 这项工作将提供 ATP结合盒成员调节离子通道的模型 超家族

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.