Dynamics of Acid-sensing ion channels

项目摘要 酸敏离子通道(ASICs)是细胞外pH的重要感受器，对细胞的兴奋性有重要作用 在中枢神经系统和外周神经系统中。ASIC将细胞外质子与细胞外的 打开一个钠选择孔。初步研究表明，ASIC可能是 疼痛以及中风等缺血性事件的治疗。有5种ASIC亚型可引起at 至少7个不同的通道亚基。ASIC既有同三聚体，也有异三聚体，它们的精确性质是 受航道组成的制约。我实验室的研究重点是潜在的分子机制 ASIC的功能以及这些通道在神经元中是如何微调的。我们的第一个重点是使用尖端技术 测量ASIC中构象变化的技术。使用替代捐赠者的FRET方法 利用过渡金属离子的荧光团，我们可以测量真实细胞中全长ASIC中的通道动力学。 有了这些数据，我们就可以建立ASIC如何打开、关闭和减敏的机械模型。尽管 异构体ASIC复合体在神经元中的普遍存在，对ASIC的化学计量学知之甚少 异构体或形成异构体的机理。使用一种名为SPACE的新荧光方法 强度分布分析(SPIDA)，我们将能够看到不同的ASIC异构体是如何异构化的。 以前的工作着眼于ASIC1a/ASIC2a异构体的化学计量比，但没有其他组合研究过 已经研究过了。我们的工作将提供第一次看到这些其他ASIC组合之间的异构化。 原则上，这种方法也与观察神经元内源性受体的化学计量学相兼容。 我们将开始朝着这个方向建立我们的系统。最后，我们对大分子络合物感兴趣。 离子通道形成。已知ASIC与Stomatin(Stom)蛋白家族有关。我们有 证明了Stom与ASIC3结合，将电流降低了近200倍。此外，我们还有 将Stom在ASIC3上的结合位点定位到两个关键区域。第一个是远端的C-末端和 其次是第一个跨膜区(TM1)。扩展这项工作，我们计划使用膜片钳 电生理学以确定ASIC3的Stom依赖调节机制。另外，我们希望 将这项工作扩展到包括Stomatin-like Protein 3(STOML3)在内的Stom家族的其他成员。 总体而言，这些研究将为ASIC在结构和细胞两个方面的功能提供新的见解 级别。

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.