3D structure and function of Acid Sensing Ion channels

 描述(申请人提供)：在大脑中，许多病理生理事件伴随着细胞外环境的酸化。不足为奇的是，在细胞外pH的这种变化期间，神经元经常表现出强大的电生理反应。酸敏感离子通道(ASIC)介导了这些电流的绝大多数，在过去的几年里，我们对ASIC神经生物学的了解有了实质性的增长。由于这项研究，ASIC与中风后的细胞死亡、疼痛、恐惧相关的记忆和药物成瘾等疾病有关。然而，我们还没有看到我们对ASIC如何在分子水平上运作的理解出现相应的激增。如果没有这些知识，我们就无法将我们日益增长的神经生物学意识转化为有用的治疗方法。候选人将把他在电生理学和荧光方面的专业知识与冷冻电子显微镜(Cryo-EM)的新培训结合起来，以弥合ASIC神经生物学和生物物理学之间的差距。这将通过三个目标来实现。在K相的特定目标1中，候选者将获得ASIC1a apo和钝化态的高分辨率低温EM结构。Jayaraman博士将提供新的蛋白质纯化培训，Gonzales博士将提供ASIC特定建议。Serysheva博士将提供低温电磁成像和3D重建方面的专家指导，达到候选人可以独立进行实验的水平。这些结构将提供载脂蛋白状态的第一次一瞥，并确定稳态脱敏(由pH接近7引发)是否与激活后的脱敏(pH接近5)构象相同。R阶段将从目标2开始，候选人和他的受训者将使用串联的亚基来确定激活的化学计量比，并确定亚基之间的构象变化是否一致。在目标3中，候选人和他的受训者将获得大脑中占主导地位的异构体ASIC的高分辨率冷冻-EM图像。功能信息和结构数据的这种组合将有助于实现候选人的两个近期目标，即对ASIC的基本操作做出必要的洞察，并成为ASIC门控领域的领导者。此外，这些研究将为开发具有竞争力的R01应用提供初步数据，重点放在ASIC异构体的结构和功能上。这项K99/R00奖还将使候选人更好地满足使ASIC突变或操纵的效果可预测的长期目标，并帮助推动开发更具选择性和更有用的药理药物的努力。

 DESCRIPTION (provided by applicant): In the brain, many pathophysiological events are accompanied by acidification of the extracellular environment. Unsurprisingly, neurons often show robust electrophysiological responses during such changes in extracellular pH. Acid sensing ion channels (ASICs) mediate the vast majority of these currents and the last few years have seen a substantial increase in our understanding of ASIC neurobiology. Because of this research, ASICs have been implicated in cell death following stroke, in pain, in fear related memory and in drug addiction amongst other disorders. However, we have not seen a corresponding surge in our understanding of how ASICs operate at a molecular level. Without such knowledge, we cannot translate our growing awareness of neurobiology into useful treatments. The candidate will combine his expertise in electrophysiology and fluorescence with new training in cryo-electron microscopy (cryo-EM) to bridge the gap between ASIC neurobiology and biophysics. This will be done through three Aims. During Specific Aim 1 in the K phase, the candidate will obtain high resolution cryo-EM structures of ASIC1a apo and desensitized states. New training in protein purification will be provided from Dr. Jayaraman and ASIC-specific advice from Dr. Gonzales. Dr. Serysheva will provide expert instruction in cryo-EM imaging and 3D reconstruction to a level where the candidate can perform the experiments independently. These structures will provide the first glimpse of the apo state and determine if steady-state desensitization (provoked by pH's near 7) is the same conformation as desensitization following activation (pH's near 5). The R phase will begin with Aim 2 where the candidate and his trainees will use concatenated subunits to determine the stoichiometry of activation and establish if conformational changes are concerted across subunits. In Aim 3, the candidate and his trainees will obtain high resolution cryo-EM images of heteromeric ASICs, the predominant population in the brain. This combination of functional information and structural data will help fulfill the candidates two near term goals of contributing essential insight into th fundamental operation of ASICs and emerging as a leader in the field of ASIC gating. Moreover, these studies will provide preliminary data to develop a competitive R01 application focusing on ASIC heteromeric structure and function. This K99/R00 award will also better position the candidate to satisfy the long term goals of rendering the effect of ASIC mutations or manipulations predictable and to help drive efforts to develop more selective and useful pharmacological agents.