Dynamics of Acid-sensing ion channels

项目摘要 酸敏感离子通道（ASIC）是细胞外pH的关键传感器，有助于细胞的兴奋性 在中枢和外周神经系统都有。ASIC将细胞外质子的结合与细胞外质子的结合偶联。 打开钠选择性孔。初步研究表明，ASIC可能是治疗癌症的可行目标。 治疗疼痛以及缺血性事件如中风。有5种ASIC同种型， 至少7个不同的通道子单元。ASIC形成同源三聚体和异源三聚体，它们的精确性质是 由渠道组成。我实验室的研究主要集中在 ASIC功能以及这些通道如何在神经元中微调。我们的第一个重点是使用尖端的 技术来测量ASIC中的构象变化。使用FRET方法代替供体 荧光团与过渡金属离子，我们可以在真实的细胞中测量全长ASIC中的通道动力学。 有了这些数据，我们可以建立ASIC如何打开、关闭和脱敏的机制模型。尽管 异聚体ASIC复合物在神经元中的流行，关于ASIC的化学计量学知之甚少 异聚体或异聚体形成的机制。使用一种新的荧光方法， 通过强度分布分析（SpIDA），我们将能够观察不同的ASIC同种型如何异聚化。 以前的工作已经着眼于ASIC 1a/ASIC 2a异聚体的化学计量，但没有其他组合 本文研究了我们的工作将提供这些其他ASIC组合之间的异聚化的第一个视图。 原则上，这种方法也与观察神经元中内源性受体的化学计量学相容。 我们将开始朝这个方向建立我们的系统。最后，我们感兴趣的是大分子复合物 离子通道的形成。已知ASIC与Stomatin（STOM）蛋白家族相关。我们有 证明了STOM与ASIC 3结合，并将电流降低了近200倍。另外我们有 将ASIC 3上STOM的结合位点定位于两个关键区域。第一个是远端C末端， 其次是第一跨膜结构域（TM 1）。扩展这项工作，我们计划使用膜片钳 电生理学以确定ASIC 3的STOM依赖性调节的机制。此外，我们希望 将这项工作扩展到包括STOM家族的其他成员，包括Stomatin-like protein 3（STOML 3）。 总的来说，这些研究将在两个方面提供新的见解，即ASIC如何在结构和细胞方面发挥作用。 程度.

Project Summary Acid-sensing ion channels (ASICs) are critical sensors of extracellular pH that contribute to excitability in cells in both the central and peripheral nervous system. ASICs couple the binding of extracellular protons to the opening of a sodium selective pore. Preliminary research has suggested that ASICs may be viable targets in the treatment of pain as well as ischemic events such as stroke. There are 5 ASIC isoforms that give rise to at least 7 different channel subunits. ASICs form both homo- and heterotrimers and their precise properties are governed by the channel composition. Research in my lab focuses on the molecular mechanisms underlying ASIC function and how these channels are fine tuned in neurons. Our first focus is on using cutting-edge techniques to measure conformational changes in ASICs. Using a FRET approach that replaces the donor fluorophore with a transition metal ion, we can measure channel dynamics in full-length ASICs in real cells. With these data, we can build mechanistic models for how ASICs open, close, and desensitize. Despite the prevalence of heteromeric ASIC complexes in neurons, little is known about the stoichiometry of ASIC heteromers or the mechanism of heteromer formation. Using a new fluorescence approach called spatial intensity distribution analysis (SpIDA), we will be able to look at how different ASIC isoforms heteromerize. Previous work has looked at the stoichiometry of ASIC1a/ASIC2a heteromers, but no other combination has been studied. Our work will provide the first look at heteromerization between these other ASIC combinations. In principle, this approach is also compatible with looking at stoichiometry of endogenous receptors in neurons. We will begin to build our system in that direction. Lastly, we are interested in the macromolecular complexes that ion channels form. ASICs are known to associate with the Stomatin (STOM) family of proteins. We have demonstrated that STOM binds to ASIC3 and reduces the current by almost 200-fold. In addition, we have localized the binding site for STOM on ASIC3 to two critical regions. The first is the distal C-terminus and the second in the first transmembrane domain (TM1). Extending this work, we plan to use patch clamp electrophysiology to determine the mechanism of STOM-dependent regulation of ASIC3. In addition, we hope to extend this work to include other members of the STOM family including Stomatin-like protein 3 (STOML3). Overall, these studies will provide new insights in two how ASICs function both at the structural and cellular levels.