K Channel & c-Src Signaling Complexes in Smooth Muscle

描述（由申请人提供）：钾通道是平滑肌收缩状态的关键调节剂。越来越多的证据表明，它们可能充当信号转导器，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。为了完成这一任务，K+通道必须与膜受体和细胞内信号分子紧密接近并形成大分子复合物。此外，这种联系应该是动态的，在不同的生理条件下，例如在怀孕期间性激素的影响下，容易发生变化。因此，我们的主要假设指出，K+通道定位于平滑肌（SM）中的特定微区，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。为了验证这一假设，我们将使用模型系统子宫SM，在怀孕期间和产后经历了巨大的结构和功能的重塑，和一个多学科的方法分析功能协会，本地亚细胞共定位，蛋白质-蛋白质相互作用的分子性，和激素机制导致重塑。研究将集中在电压和Ca 2+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，所有SM功能的关键调节剂。初步数据表明，在子宫肌层中：i）具有酪氨酸激酶活性的新的c-Src相关蛋白在妊娠晚期被进一步诱导，ii）c-Src、Kv4.3和小窝蛋白在洗涤剂抗性级分中共迁移并且可以被共免疫沉淀，iii）重组MaxiK羧基尾与c-Src相互作用，iv）单个肌细胞中的MaxiK/Kv4.3成簇模拟c-Src，v）c-Stc和小窝蛋白-la mRNA在妊娠期间增加四倍，vi）自发收缩性在Src激酶酪氨酸磷酸酶的控制下<->，和vii）其机械输出由新激动剂-&gt;c-Src酪氨酸激酶-&gt;MaxiK途径调节。因此，具体目的是研究：1）新的妊娠诱导的Src样蛋白的身份; 2）在SM MaxiK/Kv4.3中是否与c-Src、小窝蛋白和5-HT受体形成大分子复合物，它们的亚细胞共定位，以及在整个妊娠期间的潜在重塑; 3）MaxiK-c-Src-小窝蛋白-受体复合物的分子相互作用; 4）性激素引发的c-Src-caveolins-MaxiK/Kv4.3关联的妊娠相关变化的机制及其在产后的恢复; 5）Src对MaxiK/Kv4.3通道活性的调节作用及其机制，以及它们在自发或5-HT诱导的收缩性中的作用。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。

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.