K Channel & c-Src Signaling Complexes in Smooth Muscle

K频道

基本信息

批准号：
7215694
负责人：
ENRICO STEFANI
金额：
$ 32.96万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7215694
关键词：
Ablation Agonist Angiotensins Arteries Binding Biochemistry Biological Models Biology Boxing CAV1 gene Carbonates Caveolae Caveolins Cell membrane Cells Cellular biology Cholesterol Complement Complex Condition Confocal Microscopy Coupled Coupling Data Dependence Detergents Doctor of Philosophy Electrophysiology (science)Figs - dietary G Protein-Coupled Receptor Genes GTP-Binding Proteins Genes Goals Gonadal Steroid Hormones Growth Homologous Gene Hormonal Hormones Intestines Ion Channel Kinetics Kv4.3 channel Laboratories Lead Localized Macromolecular Complexes Maintenance Measures Mechanics Membrane Messenger RNA Molecular Molecular Biology Muscle Cells Muscle Contraction Muscle function Mutation Neurotransmitters Numbers Output Pathology Pathway interactions Peptides Phosphatidylinositols Phosphotransferases Physiological Physiology Play Postpartum Period Potassium Channel Pregnancy Probability Production Protein Tyrosine Kinase Protein Tyrosine Phosphatase Proteins Rate Rattus Receptor Signaling Recombinant Proteins Recombinants Recovery Research Personnel Resistance Resolution Rho-associated kinase Role SRC gene Scaffolding Protein Scheme Serotonin Serotonin Receptor 5-HT2A Signal Pathway Signal Transduction Signaling Molecule Site Smooth Muscle Sphingolipids Stimulus Structure Sucrose Surface System Tail Testing Transducers Translating Vascular Smooth Muscle Vesicle Work animation base caveolin 1 computerized data processing extracellular interdisciplinary approach large-conductance calcium-activated potassium channels molecular size myometrium programs protein protein interaction protein-tyrosine kinase c-src receptor research study response scaffold serotonin receptor uterine contractility vasoconstriction voltage

项目摘要

DESCRIPTION (provided by applicant): Potassium channels are key regulators of smooth muscle contractile state. Accumulating evidence suggests that they may act as signal transducers, translating extracellular stimuli by hormones, neurotransmitters and peptides, to intracellular signaling processes. To undertake this task, K+ channels must be in close proximity and forming macromolecular complexes with membrane receptors and intracellular signaling molecules. Moreover, this association should be dynamic and susceptible to change under different physiological conditions, for example, under the influence of sex hormones during pregnancy. Thus, our main hypothesis states that K+ channels localize to specific microdomains in smooth muscle (SM) and are intimately associated with receptors and signaling cascades either directly or indirectly via scaffolding proteins, and that, this association and subcellular distribution is influenced by sex hormones. To test this hypothesis, we will use as model system uterine SM that undergoes dramatic remodeling in structure and function during pregnancy and postpartum, and a multidisciplinary approach analyzing functional association, native subcellular colocalization, molecularity of protein-protein interactions, and the hormonal mechanisms leading to remodeling. Studies will focus on voltage and Ca2+-activated K+ (MaxiK, BKca) and Kv4.3 channels, 5-HT receptors, c-Src tyrosine kinase and caveolins, all critical regulators of SM function. Preliminary data indicate that, in myometrium: i) a new c-Src-related protein with tyrosine kinase activity is further induced in late pregnancy, ii) c-Src, Kv4.3 and caveolin comigrate in detergent resistant fractions and can be coimmunoprecipitated, iii) recombinant MaxiK carboxyl tail interacts with c-Src, iv) MaxiK/Kv4.3 in single myocytes are clustered mimicking c-Src, v) c-Stc and caveolin-la mRNAs are quadrupled during pregnancy, vi) spontaneous contractility is under the control of Src kinase <-> tyrosine phosphatase, and vii) its mechanical output is regulated by the new agonist ->c-Src tyrosine kinase ->MaxiK pathway. Thus, the Specific Aims are to investigate: 1) the identity of a new pregnancy-induced Src-like protein; 2) whether in SM MaxiK/Kv4.3 form macromolecular complexes with c-Src, caveolin and 5-HT receptors, their subcellular colocalization, and potential remodeling across gestation; 3) the molecular interactions of MaxiK-c-Src-caveolin-receptor comp/exes; 4) the mechanism(s) triggered by sex hormones leading to pregnancy-related changes of c-Src-caveolins- MaxiK/Kv4.3 associations, and their recovery in postpartum; and 5) the functional impact and mechanism of Src modulation of MaxiK/Kv4.3 channel activity, and their role in spontaneous or 5-HT induced contractility. These studies will increase our understanding of SM biology and may lead to better treatments of SM-related pathologies.

描述（由申请人提供）：钾通道是平滑肌收缩状态的关键调节因子。越来越多的证据表明，它们可能充当信号转换器，将激素、神经递质和肽的细胞外刺激转化为细胞内信号传导过程。为了承担这项任务，K+通道必须非常接近，并与膜受体和细胞内信号分子形成大分子复合物。此外，这种关联应该是动态的，并且容易在不同的生理条件下发生变化，例如在怀孕期间性激素的影响下。因此，我们的主要假设表明，K+ 通道定位于平滑肌 (SM) 中的特定微域，并通过支架蛋白直接或间接与受体和信号级联密切相关，并且这种关联和亚细胞分布受到性激素的影响。为了检验这一假设，我们将使用子宫 SM 作为模型系统，该系统在怀孕和产后期间在结构和功能上经历了巨大的重塑，并采用多学科方法分析功能关联、天然亚细胞共定位、蛋白质-蛋白质相互作用的分子性以及导致妊娠的激素机制。来改造。研究将集中于电压和 Ca2+ 激活的 K+（MaxiK、BKca）和 Kv4.3 通道、5-HT 受体、c-Src 酪氨酸激酶和小窝蛋白，这些都是 SM 功能的关键调节因子。初步数据表明，在子宫肌层中：i) 在妊娠晚期进一步诱导具有酪氨酸激酶活性的新 c-Src 相关蛋白，ii) c-Src、Kv4.3 和 Caveolin 在洗涤剂抗性级分中共迁移，并且可以共免疫沉淀， iii) 重组 MaxiK 羧基尾与 c-Src 相互作用，iv) 单肌细胞中的 MaxiK/Kv4.3 成簇模仿c-Src，v) c-Stc 和 Caveolin-la mRNA 在怀孕期间增加四倍，vi) 自发收缩力受 Src 激酶 <-> 酪氨酸磷酸酶控制，vii) 其机械输出受新激动剂调节 -> c-Src 酪氨酸激酶 ->MaxiK 途径。因此，具体目标是研究：1）新的妊娠诱导 Src 样蛋白的身份； 2) SM MaxiK/Kv4.3是否与c-Src、caveolin和5-HT受体形成大分子复合物、它们的亚细胞共定位以及整个妊娠期间的潜在重塑； 3) MaxiK-c-Src-caveolin-受体复合物/执行物的分子相互作用； 4) 性激素触发的机制导致 c-Src-caveolins- MaxiK/Kv4.3 关联的妊娠相关变化及其在产后的恢复； 5) Src 对 MaxiK/Kv4.3 通道活性调节的功能影响和机制，及其在自发收缩或 5-HT 诱导收缩中的作用。这些研究将增加我们对 SM 生物学的理解，并可能导致 SM 相关病理的更好治疗。