K Channel & c-Src Signaling Complexes in Smooth Muscle

描述(申请人提供)：钾通道是平滑肌收缩状态的关键调节器。越来越多的证据表明，它们可能起到信号转导的作用，通过激素、神经递质和多肽将细胞外刺激转换为细胞内信号过程。要完成这一任务，钾离子通道必须与膜受体和细胞内信号分子紧密相连，形成大分子复合体。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，这种联系应该是动态的和容易改变的。因此，我们的主要假设是，K+通道定位于平滑肌(SM)中特定的微区，并直接或间接地通过支架蛋白与受体和信号级联密切相关，这种联系和亚细胞分布受性激素的影响。为了验证这一假设，我们将使用妊娠和产后在结构和功能上经历戏剧性重塑的子宫SM作为模型系统，并使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特性，以及导致重塑的激素机制。研究将集中在电压和钙激活的K+(Maxik，BKca)和Kv4.3通道，5-羟色胺受体，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。初步研究表明，在子宫肌层中：(1)妊娠晚期进一步诱导出具有酪氨酸激酶活性的新的c-Src相关蛋白；(2)c-Src、Kv4.3和Caveolin共聚物在抗洗涤剂组分中可被免疫共沉淀；(3)重组Maxik羧基尾巴与c-Src相互作用；(4)单个肌细胞中的Maxik/Kv4.3与c-Src相似地聚集；(V)c-STC和Cavelin-1a mRNAs在妊娠期间增加4倍；vi)自发收缩能力受Src激酶和GT控制；C-Src酪氨酸激酶--MAXIK通路。因此，本研究的具体目的是：1)一种新的妊娠诱导的Src样蛋白的同源性；2)在SM Maxik/Kv4.3中是否与c-Src、小窝蛋白和5-羟色胺受体形成大分子复合体，它们的亚细胞共定位，以及跨孕期潜在的重构；3)Maxik-c-Src-小窝蛋白-受体comp/exes的分子相互作用；4)性激素引起c-Src-小窝蛋白-Maxik/Kv4.3组合的妊娠相关变化及其在产后的恢复机制(S)；5)Src对Maxik/Kv4.3通道活动的调节作用和机制，以及它们在自发性或5-羟色胺引起的收缩中的作用。这些研究将增加我们对SM生物学的了解，并可能导致更好地治疗SM相关的病理。

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.