MaxiK channel biology

描述(申请人提供)：大电导，钙激活的K+通道(Maxik，BK)是包括血管张力、神经功能和免疫在内的大量细胞功能的关键调节因子。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，引导它们到达功能部位，是了解它们功能基础的关键。因此，本研究的长期目标是确定Maxik通道转录、共(翻译后)修饰和流量的调节机制，这些机制决定了Maxik通道在正确的时间和地点的可用性和功能。我们已经了解了不同系统中Maxik通道的分子组成，以及它们通过沉默亚单位基因在动物生理中的作用，并开始掌握它们的细胞交通机制和潜在网络的信息。我们现在将测试一般假设，Maxik的孔形成亚基(Slo)基因和蛋白质具有内在序列，这些序列控制着它们的表达、载体运输和蛋白质-脂类相互作用，根据生理需要将它们定位在战略细胞间隔中。我们的初步数据表明：i.MSLO启动子区域对雌激素(E_2)作出反应，并包含潜在的激素反应序列，可能通过E_2调节通道表达；碱侧Maxik靶向可能由Slo剪接变体插入物驱动；以及SLO可以被肉豆蔻酰化。我们将使用多学科方法，特别是亲和素-Slo结构，用量子点和高分辨率共聚焦显微镜可视化单分子运动。其具体目的是：1)定位Slo启动子(S)的转录起始点(S)和功能的E2调节序列，并确定E2调节Slo转录的基因组机制(S)；2)研究Slo剪接变异体(S)在决定差异运输和靶向中的作用；以及3)研究Maxik肉豆蔻酸化的机制和位置及其功能后果。这些研究将为调节Maxik通道基因和蛋白表达和靶向的机制提供新的信息，并确定缓解心脑血管疾病的新治疗途径。

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.