Mechanisms behind the clustering of BK and calcium channels

项目摘要 大钾(BK)通道是一种非典型的激活钾通道，以其大K电导命名 通过电压和钙的协同作用。BK通道在几乎所有的器官中都有表达，在 神经、胃肠、生殖、视觉、内分泌、尿路、淋巴、骨骼肌的功能，以及 心血管系统。BK通道的激活需要钙与细胞质C- 频道的终端。因此，质膜的空间定位和靠近钙源 是调节BK通道功能的两个关键因素。我之前使用超分辨率显微镜所做的工作 证明BK通道不是均匀分布在质膜上，而是组织成 大型星系团。此外，我还演示了这些BK频道集群组织成了更高的层次 与电压门控钙通道(Cav)的排列，这是它们激活的主要钙来源。 结合电生理学和药理学，我确定这种BK-CAV共同聚集是 生理超极化电压下BK通道的激活。然而，调解的分子机制 BK通道簇的形成以及与特定CAV通道的共聚仍然是一个谜。研究领域 我的实验室现在以我的发现为基础，专注于识别BK-BK和BK-BK背后的分子机制- CAV通道聚集及聚集在BK功能微调中的作用。利用我们在以下领域的专业知识 超分辨率显微镜，定量荧光，离子通道生物物理学，和脂类代谢，我们有 确定了三个研究领域，将促进我们对BK通道背后的机制的理解 集群化。研究领域1将重点研究调节形成和维持的机制 BK-BK簇-为此，我们将系统地评估BK通道C-末端的作用以及BK-BK簇的作用 BKβ和γ辅助亚基。研究领域2将集中于BK-Cav通道协同调节的机制。 集群-为此，我们将AKAP150和RIB2确定为与两者交互的两个强有力的候选 渠道，并可以作为共同集群的基础。研究领域3将集中于一个很少被探索的主题，即 BK-Cav通道聚集中的膜脂组成-我将利用我的专业知识研究 磷脂酰肌醇和胆固醇对离子通道的调节作用 脂类参与了离子通道聚集。总体而言，该研究计划旨在提供新的见解 BK通道簇是如何在钙源附近形成和维持的。从长远来看，这项提议 这将为研究BK通道在健康和疾病中的作用机制开辟新的途径。

Project Summary Big potassium (BK) channels, named after their "Big K+" conductance, are atypical potassium channels activated synergistically by voltage and calcium. Expressed in nearly all organs, BK channels have important roles in the function of the nervous, gastrointestinal, reproductive, visual, endocrine, urinary, lymphatic, skeletal muscle, and cardiovascular systems. BK channel activation requires the allosteric binding of calcium to the cytoplasmic C- terminal of the channel. Therefore, spatial localization at the plasma membrane and proximity to calcium sources are two critical factors regulating BK channel function. My previous work using super-resolution microscopy has demonstrated that rather to be homogeneously distributed at the plasma membrane, BK channels organize into large clusters. In addition, I demonstrated that these BK channel clusters organize into a higher hierarchical arrangement with voltage-gated calcium channels (CaV), which are their primary calcium source for activation. Combining electrophysiology and pharmacology, I established that this BK-CaV co-clustering is key for the activation of BK channels at physiological hyperpolarized voltages. Yet, the molecular mechanisms that mediate BK channel cluster formation and the co-clustering with specific CaV channels remain mysterious. Research in my lab now builds on my discoveries and focus on identifying the molecular mechanisms behind BK-BK and BK- CaV channel clustering and the role of clustering in the fine-tuning of BK function. Leveraging our expertise in super-resolution microscopy, quantitative fluorometry, ion channel biophysics, and lipid metabolism, we have identified three research areas that will advance our understanding of the mechanisms behind BK channel clustering. Research area 1 will focus on the study of the mechanisms mediating the formation and maintenance of BK-BK clusters – for this, we will systematically evaluate the role of the BK channel C-terminal and the role of BK β and γ auxiliary subunits. Research Area 2 will focus on the mechanisms mediating BK-CaV channel co- clustering – for this, we have identified AKAP150 and RIB2 as two strong candidates that interact with both channels and can underlie the co-clustering. Research Area 3 will focus in a poorly explored topic, the role of membrane lipid composition in the clustering of BK-CaV channels – I will leverage my expertise in the study of ion-channel modulation by phosphoinositides and cholesterol to lead the research on how these two signaling lipids are involved in ion channel clustering. Overall, this research program is designed to provide new insights into how BK channel clusters are formed and maintained near calcium sources. In the long-term, this proposal will open new avenues for the study of mechanisms modulating BK channel function in health and disease.