REGULATION OF UTERINE SMOOTH MUSCLE EXCITABILITY

早产只占所有分娩的10%，但与80%以上的新生儿死亡和95%以上的主要新生儿发病率有关。对调节子宫收缩的机制缺乏了解，阻碍了有效治疗这一生殖健康问题的进展。目前使用的卵裂解剂在延长妊娠方面效果不佳，需要研究其他预防机制。一类潜在的治疗靶点是钾通道，因为它们有能力有效地缓冲细胞兴奋。电生理研究表明，子宫肌层细胞含有多种类型的K+通道，可能是潜在的治疗靶点，包括大电导钙激活的K+通道(BKCa通道)。阻断BKCa通道可使子宫肌层细胞去极化并增加收缩活动，而激动剂和β-肾上腺素能药物激活这些通道则可诱导强大的子宫松弛。有趣的是，磷酸化试剂、钙离子或电压对BKCa通道的激活依赖于它是从非妊娠组织还是妊娠组织中分离出来的。最近的证据表明，该通道在应激激素存在的情况下进行选择性剪接的能力，产生了不同的变体，它们对细胞内钙离子和电压的敏感性不同。对这些药物的敏感性也依赖于BKCa通道与其辅助的β亚基的联系。这表明，选择性剪接或调节β亚基结合可能是调节妊娠期间子宫平滑肌BKCa通道多样性的机制。虽然这一通道似乎是子宫兴奋性的重要调节成分，但它在妊娠期间调节子宫肌层收缩的作用仍不清楚。该建议的具体目的是确定BKCa通道剪接变异体表达或β亚基关联的调节是否与妊娠期子宫兴奋性的功能差异有关。BKCa通道剪接变异表达和功能的变化将通过结合小鼠的电生理和收缩测量的分子特征来研究。本研究的具体目的是：1)比较BKCa通道异构体在妊娠小鼠子宫平滑肌中的转录和蛋白表达模式；2)阐明BKCa通道β亚基在妊娠期间的转录和蛋白表达，并确定其与α亚基的组装在妊娠期间是否受到调节；3)确定BKCa通道剪接变异体在妊娠期间对子宫平滑肌收缩的调节作用；以及4)表征在雌激素和孕酮刺激下BKCa通道α亚基剪接变异体的表达。早产是一个主要的健康问题，特别是考虑到它带来的出生缺陷风险和与早产相关的成本。因此，BKCa通道是否可能成为未来药物治疗的靶点是一个值得研究的重要问题。

Preterm births account for only 10 percent of all deliveries, but are associated with over 80 percent of newborn deaths and more than 95 percent of major newborn morbidity. The lack of understanding of the mechansims regulating uterine contraction has hampered progress towards an effective treatment for this reproductive health problem. Currently used tocolytics have little effect in prolonging gestation, necessitating the investigation of other mechansims of prevention. One potential class of therapeutic targets is potassium channels, due to their ability to potently buffer cell excitation. Electrophysiological reports have shown that myometrial cells contain a variety of K+ channel types that may be potential therapeuric targets, including the large-conductance calcium-activated K+ channel (BKCa channel). Blockage of BKCa channels depolarizes myometrial cells and increases contractile activity while activation of these channels by agonists and beta-adrenergic agents induces potent uterine relaxation. Interestingly, the activation of the BKCa channel by phosphorylating agents, Ca2+, or voltage is dependent on whether it is isolated from non-pregnant or pregnant tissue. Recent evidence illustrates the ability of this channel to undergo alternative splicing in the presence of stress hormones, yielding variants that differ in their sensitivity to intracellular Ca2+ and voltage. Sensitivity to these agents is also dependent on the association of the BKCa channel with its ancillary beta subunit. This suggests that alternative splicing or modulation of beta subunit association may be mechanisms regulating BKCa channel diversity in uterine smooth muscle during gestation. While this channel appears to be an important regulatory component of uterine excitability, its role in modulating myometrial contraction during gestation remains unknown. The specific objective of this proposal is to detemine whether modulation of BKCa channel splice variant expression or beta subunit association correlates to a functional difference in uterine excitability during gestation. Alterations in BKCa channel splice variant expression and function during gestation will be investigated by molecular charactization in combination with electrophysiological and contraction measurements in mice. The specific aims of this proposal are to: 1) compare transcript and protein expression patterns of BKCa channel isoforms in mouse uterine smooth muscle during gestaiton, 2) elucidate BKCa channel beta subunit transcript and protein expression during gestation and detemine whether its assembly with the alpha subunit is modulated during pregnancy, 3) determine the contribution of BKCa channel splice variants to the regulation of uterine smooth muscle contraction during gestation, and 4) characterize the expression of the splice variants of the BKCa channel alpha subunit following stimulation with estrogen and progesterone. Preterm labor is a major health problem, especially given the risks it carries with respect to birth defects and the costs associated with premature delivery. Whether the BKCa channel could be a future target for tocolytic drug therapy is thus a significant question to examine.