Functional and molecular identification of TREK-1 channel in myometrium in relati

描述(申请人提供)：在包括人类在内的大多数物种中，怀孕期间血浆17β-雌二醇和孕酮水平升高。伴随着妊娠晚期的女性类固醇激素的升高与子宫肌层平滑肌的电重构有关。这些观察表明，女性类固醇激素可以影响与子宫平滑肌兴奋性有关的离子电导的活性和/或表达。虽然有报道称分娩机制与女性激素水平下降有关，但从离子通道表达的角度来研究分娩收缩的机制还不清楚。在怀孕期间，子宫肌层的质量急剧增加，宫内压力只有轻微的增加。要做到这一点，子宫肌层的平滑肌肉必须在怀孕期间保持放松。因此，质膜拉伸激活的K+电导可能是子宫肌源性反应的重要组成部分。多个K+通道参与子宫肌层静息膜电位和动作电位复极的调节。最近有报道称，独特的K+通道由四个跨膜片段和两个孔结构域(K2P)组成。在K2P通道中，Trek-1、Trek-2和TRAAK具有独特的功能特性，是第一个被克隆的牵张激活的K+通道。以前的研究表明，女性类固醇激素影响一些K+通道的转录表达。目标1，我们将利用膜片钳方法鉴定SDK通道在小鼠子宫肌层中的功能表达，并将表征一氧化氮对这些通道的调节及其细胞内信号机制。目的2，我们将使用常规微电极记录、等长力测量和钙离子成像分析来表征完整子宫肌层的牵张依赖性超极化和松弛。目的3，我们将利用分子和蛋白质技术研究Trek-1在小鼠子宫肌层中的表达及其与天然SDK通道的关系。目的研究未妊娠、妊娠、产后及去卵巢小鼠模型中TREK-1表达的变化，以了解不同雌性激素水平下天然SDK通道的功能表达。特别是，这项研究将扩展雌激素调节Trek-1通道的分子机制，导致不同水平的子宫肌层顺应性。了解这一机制可能允许临床干预调节分娩和分娩，减少早产和后续疾病的数量。与公共卫生相关：在过去20年里，早产率增长了30%以上。在所有早产病例中，约有40%的早产原因不明。该提案中概述的实验旨在评估伴随女性类固醇激素水平急性和慢性变化的子宫Trek-1通道的变化，以研究与怀孕和分娩机制的潜在功能关系。

DESCRIPTION (provided by applicant): In most species, including humans, plasma levels of 17beta-estradiol and progesterone increase during pregnancy. The elevation of female steroid hormones that accompanies the late stages of pregnancy has been linked to electrical remodeling of myometrial smooth muscle. These observations suggest that female steroid hormones can influence the activity and/or expression of ionic conductances involved in uterine smooth muscle excitability. Although it has been reported that the delivery mechanism is related to a decrease in female hormone levels, the mechanism of labor contraction has not been clearly studied in terms of ion channel expression. During pregnancy, the mass of myometrium increases dramatically, with only minimal increases in intrauterine pressure. To accomplish this, the smooth muscle of the myometrium must remain relaxed during pregnancy. Therefore, K+ conductances activated by stretch of the plasma membrane may contribute an important component of the myogenic response in the uterus. Several K+ channels participate in the regulation of resting membrane potential and repolarization of action potentials in the myometrium. Recently unique K+ channels have been reported that consist of four transmembrane segments and two-pore domains (K2P). Among K2P channels, TREK-1, TREK-2 and TRAAK have unique functional properties and represent the first cloned stretch-activated K+ channels. Previous studies suggest that female steroid hormones influence the transcriptional expression of a number of K+ channels. We will address the following specific aims in this proposal: Aim 1, we will identify SDK channel functional expression in the murine myometrium using patch-clamp methods and will characterize the regulation of these channels by nitric oxide and its intracellular signaling mechanisms. Aim 2, we will characterize stretch-dependent hyperpolarization and relaxation in intact myometrium using conventional microelectrode recordings, isometric force measurements and Ca2+ imaging analysis. Aim 3, we will use molecular and protein techniques to investigate TREK-1 expression in murine myometrium in relation to native SDK channels. Aim 4, we will characterize changes in TREK-1 expression in non-pregnant, pregnant, postpartum and ovariectomized murine models to understand the functional expression of native SDK channels under various levels of female hormones. In particular this investigation will expand the molecular mechanism of estrogen regulation of TREK-1 channels that leads to the different levels of myometrial compliance. Understanding this mechanism may allow for clinical intervention in the modulation of labor and delivery, reducing the number of premature births and subsequent disorders. PUBLIC HEALTH RELEVANCE: The rate of premature birth has grown by more than 30 percent in the last 20 years. In about 40 percent of all cases of preterm birth, the causes of preterm labor are unknown. Experiments outlined in this proposal are designed to evaluate changes in uterine TREK-1 channels that accompany acute and chronic changes in female steroid hormone levels to study a potential functional relationship with pregnancy and delivery mechanisms.