Conformational rearrangements underlying ASIC1a gating

描述（由申请人提供）：上皮钠通道/变性蛋白（ENaC/Deg）离子通道家族由参与机械感觉、疼痛感觉、细胞外液体积和气道表面液体体积调节的蛋白质组成。ENaC/Deg通道由不同的刺激门控，包括神经肽，机械力，细胞外质子，其中一些是组成型活性的，如ENaC。这些通道可能组织为同源或异源三聚体，并且由具有类似拓扑结构的亚基组成，所述亚基具有两个跨膜结构域（TM），所述跨膜结构域通过具有短胞内N-和C-末端的大胞外域连接。最近报道了ENaC/Deg通道的细胞外和跨膜结构域的第一个高分辨率结构，即原鸡酸敏感离子通道1（cASIC 1）。ASIC是在哺乳动物中枢和外周神经系统的神经元中表达的质子门控通道，其以静息、功能不活跃状态存在，但在细胞外酸化后经历快速激活和脱敏。cASIC 1具有杯状形状，具有从膜平面突出的大的细胞外结构域。细胞外区域被组织在称为“手掌、指关节、2球、手指和拇指”的离散亚结构域中。TM和细胞外结构域之间的过渡构成腕部。虽然晶体结构提供了cASIC 1在脱敏状态下的大量分子细节，但ASIC和其他ENaC/Deg通道的门控机制在很大程度上仍然不确定。该提案的第一个具体目标将定义与激活和脱敏相关的小鼠ASIC 1a腕和孔中的构象重排。在第二个具体的目标，我们将解决有关的拇指和手掌子域的激活，脱敏和脱敏恢复的作用的问题。将通过电压钳荧光测定法研究细胞外酸化后通道受限区域中发生的构象变化，电压钳荧光测定法是一种提供与特定门控步骤相关的局部蛋白质运动信息的技术。取代的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关于小鼠ASIC 1a门控和脱敏的潜在机制的基本问题。从这项工作中获得的信息将是有价值的理解Deg/ENaC通道调节的分子机制。 公共卫生相关性：离子通道的上皮钠通道/变性蛋白（ENaC/Deg）家族由与机械感觉、疼痛感觉、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。这些通道已经进化成具有同源结构的感觉各种各样的刺激。从这项工作中获得的信息将是有价值的，以了解这些通道的调节的分子机制。

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.