Functional consequences of heteromeric assembly of auxiliary subunits in BK chann

描述(由申请人提供)：BK通道故障与多种病理有关，如高血压、癫痫、哮喘、听力损失和肿瘤生长调节。BK通道成孔亚基几乎普遍表达，其同源四聚体是BK通道的最小功能单元。BK通道电流在不同组织中表现出相当大的表型多样性，对BK通道作为治疗靶点的潜在用途提出了挑战。辅助亚单位是组织特异性表达的蛋白质，在确定BK通道的功能特性方面发挥着重要作用。4种亚基(1-4)和最近的2种亚基(1-2)被描述为BK辅助亚基，每一种亚基都定义了复合体的生理关键性质，包括表观的钙敏感性、失活能力、激活/失活动力学和药理学。每种类型的辅助亚单位产生不同影响的事实提出了在各个通道中辅助亚单位异构体组装的功能和后果的问题，但这种可能性在BK通道中从未被探索过。在这里，我假设不同类型的辅助亚基可以在单个BK通道中共同组装，并可能对在天然细胞中观察到的BK功能多样性做出贡献。我的初步实验表明，可以出现含有？2+？3a和？2+？1的异构体通道。但是，它们共同组装的可能化学计量比是什么？这些渠道有没有独特的、或许是意想不到的功能后果？它们存在于天然组织中吗？这些问题将结合功能(单通道记录)和光学方法(单分子TIRF)来解决。单通道记录的分析将利用由单个BK通道上的每个辅助子单元赋予的独特行为。将在异种系统(非洲爪哇卵母细胞)以及WT和2-KO小鼠犁鼻器的分离细胞中研究~2+？3a和？2+？1的组合。无论这些实验的结果如何，这些结果都有望为异构体辅助亚基组装所产生的BK通道的多样性提供新的见解。 公共卫生相关性：BK通道故障与高血压和癫痫等病理疾病有关。了解BK通道的表型多样性对于评估BK通道作为潜在的治疗靶点至关重要。本项目研究将确定辅助亚基产生的BK功能多样性的新机制。

DESCRIPTION (provided by applicant): BK channel malfunction has been implicated in several pathologies such as hypertension, epilepsy, asthma, hearing loss, and tumor growth regulation. The BK channel pore-forming ¿-subunit is almost ubiquitously expressed and the homotetramer of ¿-subunits is the BK channel minimal functional unit. BK channel currents exhibit substantial phenotypic diversity among different tissues challenging the potential use of BK channels as therapeutic targets. Auxiliary subunits, which are tissue-specific expressed proteins, play a major role in defining the functional properties of BK channels. 4 types of ¿-subunits (¿1-¿4) and recently 2 types of ?-subunits (?1-?2) have been described as BK auxiliary subunits, each one defining physiologically critical properties of the complex including apparent Ca2+-sensitivity, ability to inactivate, activation/deactivation kinetics and pharmacology. The fac that each type of auxiliary subunit produces different effects raises the question of the functiona consequences of heteromeric assembly of auxiliary subunits in individual channels, but this possibility has never been explored in BK channels. Here, I hypothesize that distinct types of auxiliary subunits can coassemble in a single BK channel and potentially contribute to the BK functional diversity observed in native cells. My preliminary experiments suggest that heteromeric channels containing ¿2+¿3a and ¿2+?1 can occur. But, what are the possible stoichiometries of their coassembly? Are there unique, maybe unexpected functional consequences from such channels? Are they present in native tissues? These issues will be addressed combining functional (single channel recordings) and an optical method (single molecule TIRF). The analysis of single channel recordings will take advantage of unique behavior conferred by each of the auxiliary subunits on a single BK channel. The combinations ¿2+¿3a and ¿2+?1 will be studied in heterologous systems (Xenopus oocytes) and in isolated cells from Vomeronasal Organ of WT and ¿2-KO mice. Whatever the outcome of these experiments, the results are expected to provide new insight into the diversity of BK channels that can arise from heteromeric auxiliary subunit assembly. PUBLIC HEALTH RELEVANCE: BK channel malfunction has been implicated in pathologies such as hypertension and epilepsy. Understanding BK channel phenotypic diversity is critical for assessing BK channels as potential therapeutic targets. This project research will identify new mechanisms of BK functional diversity arising from auxiliary subunits.