Conformational rearrangements underlying ASIC1a gating

描述(申请人提供)：上皮性钠通道/退行性变蛋白(ENaC/Deg)家族离子通道由与机械感觉、痛觉、细胞外液容量和呼吸道表面液量调节有关的蛋白质组成。ENAC/Deg通道受多种刺激的调控，包括神经肽、机械力、胞外质子，其中一些是结构性激活的，如ENaCs。这些通道可能被组织成同源或异三聚体，并由具有相似拓扑结构的亚基组成，两个跨膜结构域(TM)由一个大的胞外结构域连接，具有较短的细胞内N-末端和C-末端。最近报道了第一个ENaC/Deg通道胞外区和跨膜区的高分辨结构，即鸡的鸡酸敏离子通道1(CASIC1)。ASIC是一种质子门控通道，在哺乳动物中枢和外周神经系统的神经元中表达，处于静息、功能不活跃的状态，但在细胞外酸化后经历快速激活和脱敏。CASIC1呈圣杯状，膜平面上有一个较大的胞外区突起。胞外区被组织成离散的子域，称为“手掌、指节、2-球、手指和拇指”。TMS和胞外结构域之间的过渡构成了腕部。虽然晶体结构提供了cASIC1在不敏感状态下的大量分子细节，但ASICs和其他ENaC/Deg通道的门控机制在很大程度上仍不清楚。这项提议的第一个具体目标将定义与激活和脱敏相关的小鼠ASIC1a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亚域在激活、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法进行研究，这是一种提供与特定门控步骤相关的局部蛋白质运动的信息的技术。取代半胱氨酸可及性方法将用于表征封闭和钝化状态下特定区域的结构。本申请中提出的研究解决了关于小鼠ASIC1a门控和脱敏的基本机制的基本问题。这项工作所获得的信息将对理解DeG/ENaC通道调节的分子机制有价值。 与公共健康相关：上皮性钠通道/退行性变蛋白(ENaC/Deg)家族离子通道由与机械感觉、痛觉、细胞外液体容量和呼吸道表面液体容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各种刺激。从这项工作中获得的信息将对理解这些通道的分子调控机制有价值。

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.