Song Kai, Zhong Xing-Guo, Xia Xian-Ming, Huang Jun-Hao, Fan Yi-Fei, Yuan Ren-Xiang, Xue Nai-Rui, Du Juan, Han Wen-Xiu, Xu A-Man, Shen Bing
Department of Physiology, School of Basic Medicine, Anhui Medical University, Hefei, Anhui, 230032, China; Department of Gastrointestinal Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China.
Department of Surgery, Anhui Provincial Corps Hospital of Chinese People's Armed Police Force, Heifei, Anhui, 230041, China.
Biochem Biophys Res Commun. 2015 Oct 23;466(3):456-62. doi: 10.1016/j.bbrc.2015.09.049. Epub 2015 Sep 11.
Orai1 is one of the key components of store-operated Ca(2+) entry (SOCE) involved in diverse physiological functions. Orai1 may associate with other proteins to form a signaling complex. In the present study, we investigated the interaction between Orai1 and small conductance Ca(2+)-activated potassium channel 3 (SK3). With the use of RNA interference technique, we found that the SOCE and its associated membrane hyperpolarization were reduced while Orai1 was knocked down by a specific Orai1 siRNA in guinea pig gallbladder smooth muscle. However, with the use of isometric tension measurements, our results revealed that agonist-induced muscle contractility was significantly enhanced after Orai1 protein was knocked down or the tissue was treated by SK3 inhibitor apamin, but not affected by larger conductance Ca(2+)-activated potassium channel inhibitor iberiotoxin or intermediate conductance Ca(2+)-activated potassium channel inhibitor TRAM-34. In addition, in the presence of apamin, Orai1 siRNA had no additional effect on agonist-induced contraction. In coimmunoprecipitation experiment, SK3 and Orai1 pulled down each other. These data suggest that, Orai1 physically associated with SK3 to form a signaling complex in gallbladder smooth muscle. Ca(2+) entry via Orai1 activates SK3, resulting in membrane hyperpolarization in gallbladder smooth muscle. This hyperpolarizing effect of Orai1-SK3 coupling could serve to prevent excessive contraction of gallbladder smooth muscle in response to contractile agonists.
Orai1是参与多种生理功能的储存式钙（Ca2+）内流（SOCE）的关键组成部分之一。Orai1可能与其他蛋白质结合形成信号复合物。在本研究中，我们研究了Orai1与小电导钙激活钾通道3（SK3）之间的相互作用。利用RNA干扰技术，我们发现在豚鼠胆囊平滑肌中，用特异性Orai1 siRNA敲低Orai1时，SOCE及其相关的膜超极化会降低。然而，通过等长张力测量，我们的结果显示，敲低Orai1蛋白或用SK3抑制剂蜂毒明肽处理组织后，激动剂诱导的肌肉收缩力显著增强，但不受大电导钙激活钾通道抑制剂iberiotoxin或中电导钙激活钾通道抑制剂TRAM - 34的影响。此外，在存在蜂毒明肽的情况下，Orai1 siRNA对激动剂诱导的收缩没有额外影响。在免疫共沉淀实验中，SK3和Orai1相互沉淀。这些数据表明，在胆囊平滑肌中，Orai1与SK3物理结合形成信号复合物。通过Orai1的Ca2+内流激活SK3，导致胆囊平滑肌膜超极化。Orai1 - SK3偶联的这种超极化作用可能有助于防止胆囊平滑肌对收缩性激动剂做出过度收缩反应。
