HORMONAL REGULATION OF MYOMETRIUM ION CHANNELS

本研究的长期目标是揭示子宫平滑肌中激素调节的离子通道表达和功能的变化，特别强调Ca2+依赖性K+通道（MaxiK），快速瞬态K+通道（Kv4.3， ITO）和l型Ca2+通道。主要的假设是，在怀孕期间，K+和Ca2+通道类型、同工型和调节亚基的差异表达有助于子宫兴奋性和收缩性发生的剧烈变化。我们的初步数据表明：1)在怀孕期间，MaxiK通道α亚基、Kv4.3 K+通道（ITO）和Ca2+通道α 1c和β 2a亚基的表达是不同的；2) RNAse保护实验（RPA）显示，MaxiK α亚基和Kv4.3通道蛋白表达的减少与mRNA水平的变化有关；3)阻断Kv4.3通道增强收缩性；4) Maxi K α亚基的一个新的剪接插入可能作为显性负表达调节因子；5)妊娠末期，MaxiK和Kv4.3通道表达水平的降低可能与转运改变有关。6)未妊娠大鼠肌层中β -雌二醇的表达降低了Kv4.3通道的表达。因此，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) maximk α亚基的哪些剪接变体存在于肌层中，它们对功能的影响是什么？c)肌层中ITO电流的分子性质是什么？d) ITO电流在子宫肌收缩性中的作用是什么？e) l型Ca2+通道（α 1c和β亚基）的分子组成是什么，它们在怀孕期间是否有差异表达？f) l型Ca2+电流在妊娠不同阶段的功能特性是什么？g) K+和Ca2+通道表达水平变化的机制是什么？哪些性激素控制通道表达？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：1)功能，蛋白表达和mRNA水平的变化，2)肌层中存在的MaxiK α亚基剪接变异体，它们的功能特性和表达，3)快速瞬态K+电流的分子性质和功能，4)α 1c Ca2+通道和调节β亚基的性质和变化。这些研究将与设计或改善病理情况的治疗方法有关，如早产和痛经。

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.