The large-conductance calcium-activated potassium channel (BK_Ca channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BK_Ca channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BK_Ca channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid–induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BK_Ca channel and potential candidates for OAB therapeutics.

中文翻译：

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新型大传导钙激活钾通道激活剂CTIBD的鉴定，表征及其对膀胱平滑肌的松弛作用

大传导钙激活钾通道（BK _Ca通道）在各种组织中表达，并参与平滑肌松弛。该通道在膀胱平滑肌细胞上高度表达，并调节控制肌肉收缩的自发动作电位的复极化阶段。为了发现BK _Ca通道的新型化学激活剂，我们使用基于细胞的荧光分析法筛选了包含8364种化合物的化学文库。含有异恶唑基苯骨架的化合物（化合物1）被确定为BK _Ca的有效活化剂。通道，并通过结构-活性关系研究进行结构优化，以获得4-（4-（4-氯苯基）-3-（三氟甲基）异恶唑-5-基）苯-1,3-二醇（CTIBD）。当将CTIBD应用于通道的细胞外侧时，通道的电导-电压关系朝负值移动，并且最大电导以浓度依赖的方式增加。CTIBD通过显着减慢通道关闭而不影响通道打开来改变通道的门控动力学。在大鼠组织和体内测试了CTIBD对膀胱肌肉松弛和排尿功能的影响。CTIBD浓度依赖性降低乙酰胆碱引起的膀胱平滑肌条收缩。在乙酸诱导的膀胱过度活动症（OAB）模型中，腹腔注射20 mg / kg CTIBD可有效恢复频繁的排尿收缩和降低排尿量，而不会影响其他膀胱功能参数。因此，我们的结果表明CTIBD及其衍生物是膀胱BK的新型化学激活剂_Ca通道和OAB治疗药物的潜在候选药物。