Studies of Sulfonylurea Receptors and KATP Channels

描述(由申请人提供)：该计划的长期目标是了解核苷酸和药物对ATP敏感的钾KATP通道进行调节的基础。KATP通道将膜电活动与细胞能量代谢结合起来，在从控制葡萄糖刺激的胰岛素释放到维持血管平滑肌张力等生理过程中起着关键的调节作用。β细胞KATP通道的丧失是先天性高胰岛素血症的一个原因，而磺脲类药物如甲苯磺丁胺和格列本脲是治疗NIDDM的主要药物。磺脲类受体SURS与KATP通道的KIR亚基形成孔相关，并对其进行调节。过去的工作已经建立了KATP通道的化学计量学，(KIR6.x/SUR)4，并确定了指定通道异构体差异的SUR结构域，包括那些对通道阻断剂和开放剂的作用至关重要的结构域。在没有SURS的情况下，同分KIR6.2孔具有低的开放通道概率POmax，对磺脲类不敏感，表现出改变的爆裂，被ATP抑制得很差，也不被镁ADP刺激，所有这些特性都被与SUR共组装逆转。最近的工作表明，第一组跨膜螺旋TMD0和SUR的连接片段L0和KIR6.x的氨基末端对于控制慢速门控至关重要。这些片段与格列本脲的类似物亲和标记，证明它们非常接近。已经确定了对SUR-KIR组装至关重要的SUR结构域。TMD0与KIR相互作用，增加它们的Pomax，而L0以双向方式调制门控。这项研究的目的是确定参与组装和调节KATP通道的分子间和分子内相互作用。我们有三个具体的目标：(1)定义与KIR6.2相互作用所需的TMD0的最小片段，并确定这是否足以激活(KIR6.2deltaC)4孔。(2)验证KIR6.x的氨基末端与SURS的L0连接子相互作用以控制猝发的假设。(3)进一步确定SUR1的磺酰脲/格列本脲结合口袋，特别是鉴定靠近甲格列汀头基团的氨基酸。

DESCRIPTION (provided by applicant): The long-term goal of this program is understanding the basis for regulation of ATP-sensitive potassium KATP channels by nucleotides and pharmacologic agents. KATP channels couple membrane electrical activity to cellular energy metabolism and are key regulators in physiologic processes ranging from control of glucose stimulated insulin release to maintenance of vascular smooth muscle tone. Loss of beta-cell KATP channels is a cause of congenital hyperinsulinism, while sulfonylureas like tolbutamide and glibenclamide, are a mainstay in the treatment of NIDDM. Sulfonylurea receptors, SURs, associate with and regulate the pore forming KIR subunits of KATP channels. Past work has established the stoichiometry of KATP channels, (KIR6.x/SUR)4, and identified SUR domains which specify channel isoform differences including those critical for the action of channel blockers and openers. Without SURs, homomeric KIR6.2 pores have a low open channel probability, POmax, are insensitive to sulfonylureas, display altered bursting, are poorly inhibited by ATP, and not stimulated by MgADP, all properties reversed by co-assembly with an SUR. Recent work demonstrates that the first set of transmembrane helices, TMD0, and a connecting segment of SUR, L0, and the amino terminus of KIR6.x, are critical for the control of slow gating. These segments affinity-label with analogues of glibenclamide arguing for their close proximity. The SUR domain critical for SUR-KIR assembly has been identified. TMD0 interacts with KIRS increasing their Pomax while L0 modulates gating in a bi-directional manner. The goal of the proposed research is to define the inter- and intramolecular interactions involved in the assembly and regulation of KATP channels. We have three specific aims: (1) To define the minimal segment of TMD0 needed to interact with KIR6.2 and determine if this is sufficient to activate (KIR6.2deltaC)4 pores. (2) To test the hypothesis that the amino terminus of KIR6.x interacts with the L0 linker of SURs to control bursting. (3) To define further the sulfonylurea/glibenclamide binding pocket of SUR1, specifically to identify amino acids in proximity to the meglitinide head group.