Parajuli Shankar P, Hristov Kiril L, Cheng Qiuping, Malysz John, Rovner Eric S, Petkov Georgi V
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Coker Life Sciences Building, Room 609D, 715 Sumter St, Columbia, SC, 29208, USA.
Pflugers Arch. 2015 Apr;467(4):665-75. doi: 10.1007/s00424-014-1537-8. Epub 2014 May 28.
Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large-conductance Ca(2+)-activated K(+) (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9 ± 1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca(2+) from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1 channels were examined under conditions of removing the major cellular Ca(2+) sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca(2+), thus increasing the excitability in human DSM cells.
毒蕈碱型乙酰胆碱受体（mAChRs）的激活是增强人逼尿肌平滑肌（DSM）兴奋性和收缩性的主要机制。由于大电导钙激活钾（KCa1.1）通道是人类DSM功能的关键调节因子，我们研究了mAChR激活是否通过抑制KCa1.1通道来增加人DSM的兴奋性。我们使用mAChR激动剂卡巴胆碱，通过穿孔全细胞和单KCa1.1通道膜片钳记录，来确定从开放性膀胱手术中获取的新鲜分离的人DSM细胞在mAChR激活后KCa1.1通道活性的变化。从29名患者（23名男性和6名女性，平均年龄65.9±1.5岁）中收集人DSM细胞。卡巴胆碱抑制了KCa1.1通道介导的自发瞬时外向电流和自发瞬时超极化的幅度和频率，这些电流和超极化是由兰尼碱受体释放Ca(2+)触发的。卡巴胆碱还引起膜电位去极化，而在KCa1.1通道抑制剂iberiotoxin存在的情况下未观察到这种去极化，这表明KCa1.1通道的关键作用。在使用药理学抑制剂（毒胡萝卜素、兰尼碱和硝苯地平）去除KCa1.1通道激活的主要细胞内Ca(2+)来源的条件下，研究了卡巴胆碱对KCa1.1通道的潜在直接影响。在这些抑制剂存在的情况下，卡巴胆碱不影响单KCa1.1通道开放概率和平均KCa1.1通道电导（细胞贴附配置）或去极化诱导的全细胞稳态KCa1.1电流。这些数据支持这样的概念，即mAChR激活触发由细胞内Ca(2+)介导的间接功能性KCa1.1通道抑制，从而增加人DSM细胞的兴奋性。
