MaxiK channel biology: from transcription to proteomics

描述（由申请人提供）：大电导、Ca 2+激活的K+通道（MaxiK、BK）是多种细胞功能（包括血管张力、神经元功能和免疫力）的关键调节剂。 因此，了解它们的自然成熟步骤，从合成到与蛋白质建立相互作用，将它们引导到其功能位点，是了解其功能基础的关键。 因此，本研究的长期目标是确定MaxiK通道转录，共（后）-翻译修饰和交通的调控机制，以确定其在正确的时间和地点的可用性和功能。 我们已经了解了MaxiK通道在不同系统中的分子组成，它们通过沉默亚基基因在动物生理学中的作用，并开始掌握有关其细胞交通机制及其潜在网络的信息。 我们现在将测试一般假设，即MaxiK的成孔亚基（Slo）基因和蛋白质具有内在序列，这些序列控制它们的表达、矢量交通和蛋白质-脂质相互作用，根据生理需要将它们定位在战略细胞隔室中。 我们的初步数据表明：i。 mSlo启动子区响应于雌激素（E2）并且含有可能通过E2控制通道表达的潜在的转录响应序列; ii. 基底外侧MaxiK靶向可以由Slo剪接变体插入物驱动;和iii. Slo可以被豆蔻酰化。 我们将使用一个多学科的方法，特别是，抗生物素蛋白-SLO结构的可视化单分子运动与量子点和高分辨率共聚焦显微镜。 具体目标是：1）定位Slo启动子中的转录起始位点和功能性E2调节序列，并定义E2介导的Slo转录调节的基因组机制; 2）研究Slo剪接变体在确定差异运输和靶向中的作用;以及3）研究MaxiK豆蔻酰化的机制和位点及其功能后果。 这些研究应该提供关于调节MaxiK通道基因和蛋白表达和靶向的机制的新信息，并确定新的治疗途径以减轻心血管疾病。

DESCRIPTION (provided by applicant): Large conductance, Ca2+-activated K+ channels (MaxiK, BK) are key regulators of a plethora of cell functions including vascular tone, neuronal function, and immunity. As such, knowing their natural maturation steps from synthesis to the establishment of interactions with proteins that guide them to their functional sites is key to understand the basis of their function. Thus, the long-term goal of this research is to identify the regulatory mechanisms of MaxiK channel transcription, co(post)-translational modifications, and traffic that determine its availability and function at the right time and place. We have learned about the molecular composition of MaxiK channels in different systems, their role in animal physiology by means of silencing subunit genes, and have started to grasp information on the mechanisms of their cellular traffic and on their potential networks. We will now test the general hypothesis that, MaxiK's pore-forming a subunit (Slo) gene and protein have intrinsic sequences that rule their expression, vectorial traffic, and protein-lipid interactions localizing them in strategic cell compartments according to physiological needs. Our preliminary data indicate that: i. mSlo promoter region responds to estrogen (E2) and contains potential hormone-response sequences that may rule channel expression by E2; ii. basolateral MaxiK targeting may be driven by a Slo splice variant insert; and iii. Slo can be myristoylated. We will use a multidisciplinary approach, in particular, avidin-Slo constructs for visualization of single-molecule movements with quantum dots and high resolution confocal microscopy. Specific Aims are to: 1) map the transcription start site(s) and functional E2-regulatory sequences in Slo promoter(s), and define the genomic mechanism(s) of E2-mediated regulation of Slo transcription; 2) investigate the role of Slo splice variant(s) in determining differential trafficking and targeting; and 3) investigate the mechanism and site of MaxiK myristoylation and its functional consequences. These studies should provide new information on the mechanisms that regulate MaxiK channel gene and protein expression and targeting, and identify new therapeutic pathways to alleviate cardio- or cerebro-vascular diseases.