Structural basis of KATP Channel Gating

描述（由申请人提供）：atp敏感钾（KATP）通道将细胞代谢与膜兴奋性结合起来。它们通过引发与代谢信号相适应的生理反应，在能量稳态中起着至关重要的作用。KATP通道是内纠偏钾（Kir）通道家族的一员，其独特之处是需要Kir6.2与磺酰脲受体（SUR）（一种ABC转运体）共同组装才能实现功能表达。在胰腺细胞中，Kir6.2和SUR1形成的KATP通道介导葡萄糖刺激的胰岛素分泌。降低通道功能的Kir6.2或SUR1突变导致先天性高胰岛素血症，而增加通道功能的突变导致新生儿糖尿病。Kir6.2和SUR1在通道复杂的门控调节中都起着不可或缺的作用。该项目的长期目标是了解通道蛋白在门控方面的结构-功能关系。上一周期的工作已经确定并阐明了孔隙形成亚基Kir6.2在通道门控中的几个结构特征。在这个更新应用中，我们将讨论SUR1和Kir6.2之间功能耦合的机制。SUR1增加Kir6.2的开放概率（Po），使Kir6.2对ATP的抑制作用和膜磷肌苷的刺激作用超敏，并赋予MgADP以及磺脲类和二氮氧化合物对Kir6.2的药理作用。因此，SUR1和Kir6.2之间的功能耦合对于通道门控至关重要；然而，关于SUR1对Kir6.2多重影响的机制仍然存在重大的知识空白。本更新申请的目的是阐明SUR1和Kir6.2之间功能耦合的机制和结构基础。基于我们的初步数据，我们提出了一个统一的假设来解释SUR1如何对Kir6.2门控施加多重影响。具体来说，我们假设SUR1通过将Kir6.2中的TMD0短胞质环残基与Kir6.2的n端结构域残基之间的分子相互作用稳定在pip2结合的开放状态，从而赋予KATP通道固有的Po；ATP、MgADP和药物通过改变SUR1-Kir6.2界面来增强或减弱通道与pip2的相互作用，进而调节通道活性。我们将采用疾病突变、引导诱变筛选、化学交联和结构建模方法来交织前向遗传学方法来检验假设。这项研究具有创新性，因为它为该领域提出了一个新的概念。从人类健康和基础科学的角度来看，这项研究都具有重要意义。它将确定新的疾病机制，直接促进几种罕见但破坏性的婴儿/儿童疾病的诊断和治疗，并将导致更好地理解通道的结构-功能关系，以促进如何调节通道活性以治疗由通道功能障碍引起的疾病，包括II型糖尿病的新思路。这也将有助于更好地理解沉默的ABC转运蛋白如何调节离子通道以推进ABC转运蛋白和离子通道场。

DESCRIPTION (provided by applicant): ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane excitability. They play a vital role in energy homeostasis by eliciting physiological response appropriate to metabolic signals. A member of the inwardly rectifying potassium (Kir) channel family, KATP channels are unique in requiring co-assembly of Kir6.2 with a sulfonylurea receptor (SUR), an ABC transporter, for functional expression. In pancreatic ¿- cells, KATP channels formed by Kir6.2 and SUR1 mediate glucose-stimulated insulin secretion. Mutations in Kir6.2 or SUR1 that reduce channel function cause congenital hyperinsulinism whereas those that increase channel function cause neonatal diabetes. Both Kir6.2 and SUR1 play an integral role in the channel's complex gating regulation. The long-term goal of this project is to understand the structure-function relationship of channel proteins with respect to gating. Work in the previous cycle has identified and elucidated the role of several structural features of the pore-forming subunit Kir6.2 in channel gating. In this renewal application, we will address the mechanisms of functional coupling between SUR1 and Kir6.2. SUR1 increases the open probability (Po) of Kir6.2, hypersensitizes Kir6.2 to the inhibitory effect of ATP and stimulatory effect of membrane phosphoinositides, and confers the effects of MgADP as well as the pharmacological agent sulfonylureas and diazoxide on Kir6.2. Functional coupling between SUR1 and Kir6.2 is thus essential to channel gating; yet significant knowledge gaps remain regarding the mechanisms by which SUR1 exerts its multiple effects on Kir6.2. The goal of this renewal application is to elucidate the mechanisms and structural basis that are responsible for functional coupling between SUR1 and Kir6.2. Based on our preliminary data, we propose a unifying hypothesis to explain how SUR1 imposes its multiple effects on Kir6.2 gating. Specifically, we hypothesize that SUR1 confers the intrinsic Po of KATP channels by stabilizing Kir6.2 in the PIP2-bound open state via molecular interactions between residues in the short cytoplasmic loops of TMD0 in SUR1 and those in the N-terminal domain of Kir6.2; and ATP, MgADP and pharmacological agents modulate channel activity in turn by changing the SUR1-Kir6.2 interface to strengthen or weaken channel-PIP2 interactions. We will interweave forward genetics approach employing disease mutations, guided mutagenesis screening, chemical cross-linking and structural modeling approaches to test the hypothesis. The research is innovative because it presents a novel concept to the field. The research is significant from both the human health and basic science standpoints. It will identify new disease mechanisms to directly facilitate diagnosis and treatment of several rare but devastating infant/childhood diseases and will lead to a better understanding of the structure-function relationship of the channel to foster new ideas on how to modulate channel activity to treat diseases caused by channel dysfunction, including type II diabetes. It will also lead to a better understanding of how a silent ABC transporter regulates an ion channel to advance both the ABC transporter and the ion channel fields. PUBLIC HEALTH RELEVANCE: The ATP-sensitive potassium (KATP) channels play a key role in linking metabolic signals to physiological responses in many cell types. Dysfunction of KATP channels causes human disease including diabetes, hyperinsulinism, cardiac myopathy and neurological deficits. The goal of this project is to understand the molecular basis underlying the ability of the channel to open or close according to metabolic signals to facilitate development of novel therapeutic agents for disease caused by channel dysfunction.