Conformational rearrangements underlying ASIC1a gating

ASIC1a 门控基础的构象重排

• 批准号: 8286392
• 负责人: Marcelo Daniel Carattino
• 金额: $ 31.12万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-20 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8286392
• 关键词: ASIC channel; Address; Area; Binding; Binding Sites; Chickens; Couples; Coupling; Cysteine; Cystic Fibrosis; DNA Sequence Rearrangement; Data; Epithelial; Esthesia; Extracellular Domain; Extracellular Fluid; Extracellular Structure; Family; Fingers; Fluorometry; Hand; Homo; Hypertension; Hypotension; Investigation; Ion Channel; Lung diseases; Mechanics; Membrane; Methods; Molecular; Molecular Conformation; Motion; Mus; Names; Neurons; Neuropeptides; Pain; Peripheral Nervous System; Proteins; Protons; Recovery; Regulation; Reporting; Resolution; Rest; Role; Shapes; Site-Directed Mutagenesis; Sodium Channel; Stimulus; Structure; Techniques; Thumb structure; Translating; Transmembrane Domain; Vestibule; Work; Wrist; airway surface liquid; base; desensitization; epithelial Na+ channel; extracellular; public health relevance; voltage clamp

DESCRIPTION (provided by applicant): The epithelial sodium channel/degenerin (ENaC/Deg) family of ion channels is constituted of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume and airway surface liquid volume. ENaC/Deg channels are gated by diverse stimuli including neuropeptides, mechanical forces, extracellular protons, and some of them are constitutively active, such as ENaCs. These channels are likely organized as homo- or hetero- trimers and are composed of subunits that have a similar topology with two transmembrane domains (TMs) connected by a large extracellular domain with short intracellular N- and C- termini. The first high resolution structure of the extracellular and membrane-spanning domains of an ENaC/Deg channel, Gallus gallus (chicken) acid-sensing ion channel 1 (cASIC1), was recently reported. ASICs are proton-gated channels expressed throughout neurons of mammalian central and peripheral nervous systems that exist in a resting, functionally inactive state, but undergo a rapid activation and desensitization following extracellular acidification. cASIC1 has a chalice-like shape with a large extracellular domain protruding from the plane of the membrane. The extracellular region is organized in discrete subdomains named the "palm, knuckle, 2-ball, finger and thumb". The transition between the TMs and extracellular domain constitute the wrist. While the crystal structure provides great molecular details of cASIC1 in the desensitized state, the mechanism of gating of ASICs and other ENaC/Deg channels remains largely undefined. The first specific aim of this proposal will define conformational rearrangements in the wrist and pore of mouse ASIC1a associated with activation and desensitization. In the second specific aim we will address questions regarding the role of the thumb and palm subdomains in activation, desensitization and recovery from desensitization. Conformational changes that occur in restricted areas of the channel following extracellular acidification will be investigated by voltage clamp fluorometry, a technique that provides information regarding local protein motion associated with specific gating steps. The substituted- cysteine-accessibility method will be used to characterize the structure of specific regions in the closed and desensitized states. Studies proposed in this application address fundamental questions regarding the underlying mechanism of gating and desensitization of mouse ASIC1a. Information derived from this work will be valuable to comprehend the molecular mechanisms of regulation of Deg/ENaC channels. PUBLIC HEALTH RELEVANCE: The epithelial sodium channel/degenerin (ENaC/Deg) family of ion channels is constituted of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume and airway surface liquid volume. These channels have evolved to sense a broad variety of stimuli with homologous structures. Information derived from this work will be valuable to comprehend the molecular mechanisms of regulation of these channels.

描述（由申请人提供）：离子通道的上皮钠通道/退化蛋白（ENaC/Deg）家族由与机械感觉、痛觉、细胞外液体积和气道表面液体体积的调节有关的蛋白质组成。 ENaC/Deg 通道受到多种刺激的门控，包括神经肽、机械力、细胞外质子，其中一些通道​​具有组成型活性，例如 ENaC。这些通道可能被组织为同源三聚体或异源三聚体，并由具有相似拓扑结构的亚基组成，其中两个跨膜域 (TM) 通过具有短胞内 N 端和 C 端的大胞外域连接。最近报道了 ENaC/Deg 通道细胞外和跨膜结构域的第一个高分辨率结构，即鸡内酸敏感离子通道 1 (cASIC1)。 ASIC 是在哺乳动物中枢和周围神经系统的神经元中表达的质子门控通道，这些神经元以静止、功能不活跃的状态存在，但在细胞外酸化后经历快速激活和脱敏。 cASIC1 具有圣杯状形状，具有从膜平面突出的大细胞外结构域。细胞外区域被组织成离散的子域，称为“手掌、指关节、二球、手指和拇指”。 TM 和细胞外域之间的过渡构成了腕部。虽然晶体结构提供了脱敏状态下 cASIC1 的大量分子细节，但 ASIC 和其他 ENaC/Deg 通道的门控机制在很大程度上仍不清楚。该提案的第一个具体目标是定义与激活和脱敏相关的小鼠 ASIC1a 的手腕和毛孔中的构象重排。在第二个具体目标中，我们将解决有关拇指和手掌子域在激活、脱敏和脱敏恢复中的作用的问题。细胞外酸化后通道受限区域发生的构象变化将通过电压钳荧光测定法进行研究，该技术提供与特定门控步骤相关的局部蛋白质运动的信息。取代半胱氨酸可及性方法将用于表征封闭和脱敏状态下特定区域的结构。本申请中提出的研究解决了有关小鼠 ASIC1a 门控和脱敏的基本机制的基本问题。从这项工作中获得的信息对于理解 Deg/ENaC 通道调节的分子机制非常有价值。 公共健康相关性：上皮钠通道/退化蛋白 (ENaC/Deg) 离子通道家族由与机械感觉、痛觉、细胞外液量和气道表面液体量调节有关的蛋白质组成。这些通道已经进化到能够感知具有同源结构的多种刺激。从这项工作中获得的信息对于理解这些通道调节的分子机制非常有价值。