Conformational rearrangements underlying ASIC1a gating

ASIC1a 门控基础的构象重排

• 批准号: 7992929
• 负责人: Marcelo Daniel Carattino
• 金额: $ 34.79万
• 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/7992929
• 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/脱胶蛋白（ENAC/DEG）家族由与机械敏感，疼痛感觉，细胞外流体体积和气道表面液体体积相关的蛋白质组成。 ENAC/DEG通道通过包括神经肽，机械力，细胞外质子以及其中一些具有组成型活性（例如ENAC）（例如ENAC）的多种刺激门控。这些通道可能组织为同型或异构体，由具有相似拓扑的亚基组成，这些拓扑与两个跨膜结构域（TMS）相似，由大型细胞外结构域连接，其细胞内N-和C-末端短。最近报道了ENAC/DEG通道的细胞外和膜跨膜域的第一个高分辨率结构，Gallus Gallus（Chicken）酸性离子通道1（Casic1）。 ASIC是质子门控通道，它们在整个哺乳动物中心和周围神经系统的神经元中表达，它们存在于静止的，功能性不活跃的状态，但在细胞外酸化后经历了快速的激活和脱敏。 CASIC1具有类似的圣杯状形状，其细胞外结构域与膜平面突出。细胞外区域是在离散子域中组织的，名为“棕榈，指关节，2球，手指和拇指”。 TMS和细胞外域之间的过渡构成手腕。虽然晶体结构在脱敏状态下提供了大量的casic1分子细节，但ASIC和其他ENAC/DEG通道的门控机制仍然在很大程度上不确定。该提案的第一个具体目的是定义与激活和脱敏相关的小鼠ASIC1A的腕部和孔中的构象重排。在第二个特定目的中，我们将解决有关拇指和棕榈子域在激活，脱敏和脱敏中恢复中的作用的问题。细胞外酸化后通道限制区域发生的构象变化将通过电压夹具荧光测定法研究，该技术提供了有关与特定门控步骤相关的局部蛋白质运动的信息。取代的半胱氨酸 - 访问方法将用于表征封闭和脱敏状态中特定区域的结构。本申请中提出的研究涉及有关小鼠ASIC1A的门控和脱敏的基本问题的基本问题。从这项工作中得出的信息对于理解DEG/ENAC通道调节的分子机制将很有价值。 公共卫生相关性：离子通道的上皮钠通道/脱胶蛋白（ENAC/DEG）家族由蛋白质组成，蛋白质与机械敏化，疼痛感觉，细胞外流体体积和气道表面液体体积的调节相关。这些渠道已经演变成具有同源结构的各种刺激。从这项工作中得出的信息对于理解这些通道调节的分子机制将是有价值的。