Wakle-Prabagaran Monali, Lorca Ramón A, Ma Xiaofeng, Stamnes Susan J, Amazu Chinwendu, Hsiao Jordy J, Karch Celeste M, Hyrc Krzysztof L, Wright Michael E, England Sarah K
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO 63110;
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242;
Proc Natl Acad Sci U S A. 2016 Apr 19;113(16):E2335-44. doi: 10.1073/pnas.1516863113. Epub 2016 Apr 4.
The large-conductance, voltage-gated, calcium (Ca(2+))-activated potassium channel (BKCa) plays an important role in regulating Ca(2+)signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCain myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCaand both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCain an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca(2+)to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca(2+)-free medium and inhibitors of various Ca(2+)signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca(2+)through store-operated Ca(2+)channels. Finally, we found that the specific BKCablocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCachannel in human myometrium and that BKCaand its immunomodulatory interacting partners regulate Ca(2+)dynamics in hMSMCs during pregnancy.
大电导、电压门控、钙（Ca(2+)）激活的钾通道（BKCa）在调节Ca(2+)信号传导中起重要作用，并且与孕期子宫静息的维持有关。我们使用免疫纯化和质谱法来鉴定与足月妊娠（≥37周妊娠）妇女子宫肌层样本中BKCa相互作用的蛋白质。通过该筛选，我们鉴定出α2-巨球蛋白（α2M）。然后，我们使用免疫沉淀接着免疫印迹以及邻近连接分析，以证实BKCa与α2M及其受体低密度脂蛋白受体相关蛋白1（LRP1）在培养的原代人子宫肌层平滑肌细胞（hMSMCs）中的相互作用。在细胞贴附模式下的单通道电生理记录表明，活化的α2M（α2M*）以振荡模式增加了hMSMCs中BKCa的开放概率。此外，α2M使催产素预处理的hMSMCs中的细胞内Ca(2+)水平发生振荡。振荡的起始需要α2M与LRP1之间的相互作用。通过使用无Ca(2+)培养基和各种Ca(2+)信号通路的抑制剂，我们证明振荡需要细胞外Ca(2+)通过储存操纵性Ca(2+)通道进入。最后，我们发现特异性BKCa阻滞剂哌克昔林抑制了振荡，而通道开放剂NS11021增加了这些振荡的速率。这些数据表明，α2M*和LRP1调节人子宫肌层中的BKCa通道，并且BKCa及其免疫调节相互作用伙伴在孕期调节hMSMCs中的Ca(2+)动态。
