REGULATION OF UTERINE SMOOTH MUSCLE EXCITABILITY

早产仅占所有分娩的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。由于对调节子宫收缩的机制缺乏了解，妨碍了在有效治疗这一生殖健康问题方面取得进展。目前使用的促孕药对延长妊娠的作用不大，需要研究其他预防机制。钾通道是一类潜在的治疗靶点，因为它们能够有效地缓冲细胞兴奋。电生理学报告显示，肌内膜细胞含有多种可能成为潜在治疗靶点的K+通道类型，包括大电导钙激活的K+通道（BKCa通道）。阻断BKCa通道使子宫内膜细胞去极化并增加收缩活性，而激动剂和β -肾上腺素能药物激活这些通道可诱导有效的子宫松弛。有趣的是，磷酸化剂、Ca2+或电压对BKCa通道的激活取决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，该通道在应激激素的存在下进行选择性剪接的能力，产生对细胞内Ca2+和电压敏感性不同的变体。对这些药物的敏感性也取决于BKCa通道与其附属β亚基的关联。这表明β亚基关联的选择性剪接或调节可能是妊娠期子宫平滑肌BKCa通道多样性的调节机制。虽然这一通道似乎是子宫兴奋性的重要调节成分，但其在妊娠期子宫肌收缩调节中的作用尚不清楚。本研究的具体目的是确定BKCa通道剪接变体表达或β亚基关联的调节是否与妊娠期间子宫兴奋性的功能差异相关。通过结合小鼠电生理和收缩测量的分子表征，研究妊娠期间BKCa通道剪接变异表达和功能的变化。这项建议的具体目的是：1)比较妊娠期小鼠子宫平滑肌BKCa通道亚型的转录和蛋白表达模式；2)阐明妊娠期BKCa通道β亚基转录和蛋白表达，确定其与α亚基的组装是否在妊娠期被调节；3)确定BKCa通道剪接变异体对妊娠期子宫平滑肌收缩的调节作用。4) BKCa通道α亚基剪接变异体在雌激素和孕激素刺激下的表达特征。早产是一个重大的健康问题，特别是考虑到早产带来的出生缺陷风险和与早产有关的费用。因此，BKCa通道是否可能成为未来溶胎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.