HORMONAL REGULATION OF MYOMETRIUM ION CHANNELS

本研究的长期目标是揭示激素对子宫平滑肌细胞离子通道表达和功能的调节作用，特别是钙依赖性钾通道(Maxik)、快速瞬时钾通道(Kv4.3、Ito)和L型钙通道。主要的假说是，在怀孕期间，钾和钙离子通道类型、异构体和调节亚基的不同表达导致了子宫兴奋性和收缩能力的戏剧性变化。我们的初步数据表明：1)Maxik通道α亚基，Kv4.3 K+通道(ITO)，以及钙通道α1C和β2a亚基的表达在妊娠期间变化；2)RPA分析表明，Maxikα亚基和Kv4.3通道蛋白表达的降低与mRNA水平的变化有关；3)阻断Kv4.3通道增强了收缩能力；4)Maxi Kα亚基的一个新的剪接插入可能是一个显性的负表达调节因子；5)妊娠末期Maxik和Kv4.3通道表达水平的降低可能与子宫运输的改变有关；6)非妊娠大鼠子宫肌层经β-雌二醇刺激后，Kv4.3通道表达降低。因此，需要回答的问题是：a)Maxik通道在怀孕期间经历了哪些生理和药理学变化？B)在子宫肌层中存在哪些Maxikα亚基的剪接变体，它们对功能有什么影响？C)子宫肌层ITO电流的分子性质是什么？D)ITO电流在子宫肌层收缩中的作用是什么？E)L型钙离子通道(α1C和β亚基)的分子组成是什么，它们在妊娠期间是否存在差异表达？F)妊娠不同阶段L钙电流的功能特点是什么？G)钾、钙通道表达水平变化的机制是什么(S)，又是哪种性激素(S)控制通道表达？具体目标将使用多学科方法来研究怀孕不同阶段和激素治疗的：1)Maxik通道的功能、蛋白表达和mRNA水平的变化，2)在子宫肌层中存在哪些Maxikα亚基剪接变异体，它们的功能特性及其表达，3)快瞬变K+电流的分子性质和功能，以及4)Alpha1C钙通道和调节性β亚基的性质和变化。这些研究将与设计或改进治疗早产和痛经等病理情况的治疗方法(S)相关。

The long term goal of this proposal is to unravel hormonal-regulated changes of ion channel expression and function in uterine smooth muscle, with special emphasis on Ca2+ dependent K+ channels (MaxiK), fast transient K+ channels (Kv4.3, ITO) and L-type Ca2+ channels. The main hypothesis is that during pregnancy differential expression of K+ and Ca2+ channel types, isoforms, and regulatory subunits contribute to the dramatic changes that occur in uterine excitability and contractility. Our preliminary data show that: 1) Expression of the MaxiK channel alpha subunit, the Kv4.3 K+ channel (ITO), and the Ca2+ channel alpha1C and beta2a subunits, varies during pregnancy; 2) RNAse protection assay (RPA) shows that the reduction in protein expression of MaxiK alpha subunit and Kv4.3 channels correlates with changes in mRNA levels; 3) Blockade of Kv4.3 channels enhances contractility; 4) A novel splice insert of the Maxi K alpha subunit may act as a dominant negative expression regulator; 5) Reduction in the expression level, at the end of pregnancy, of MaxiK and Kv4.3 channels may be associated with altered trafficking, and 6) Myometrium from non-pregnant rats primed with beta-estradiol have reduced Kv4.3 channel expression. Thus, the questions to answer are: a) What are the physiological and pharmacological changes that MaxiK channels undergo during pregnancy? b) Which splice variants of MaxiK alpha subunit are present in myometrium and what is their functional impact? c) What is the molecular nature of ITO currents in myometrium? d) What is the role of ITO currents in myometrial contractility? e) What are the molecular components of L-type Ca2+ channels (alpha1C and beta subunits), and are they differentially expressed during pregnancy? f) What are the functional properties of L-type Ca2+ currents at different stages of pregnancy? g) Which is the mechanism(s) responsible for the changes in expression levels of K+ and Ca2+ channels, and which sex hormone(s) controls channel expression? The Specific Aims will use a multidisciplinary approach to investigate at different stages of pregnancy and with hormonal treatment: 1) changes in function, protein expression and mRNA levels of the MaxiK channel, 2) which MaxiK alpha subunit splice variants are present in myometrium, their functional properties, and their expression, 3) the molecular nature and function of fast transients K+ currents, and 4) the nature and changes in alpha1C Ca2+ channels and regulatory beta subunits. These studies will be relevant to design or improve therapeutic treatment(s) for pathological situations such as premature labor and dysmenorrhea.