STREX调控BKCa通道活性参与Oddi括约肌功能紊乱的机制研究

Sphincter of Oddi dysfunction (SOD) is regarded as the common cause for the dysfunctions of liver, gallbladder and pancreas and the clarification of the underlying mechanism for SOD play an important role in the clinical settings. The key reason for SOD is the impaired relaxation and constriction of sphincter of Oddi (SO) that may be caused by the abnormal mechanism that the larger conductance Ca2+-activated potassium channel (BKCa) can’t normally inhibit the overload of intracellular calcium concentration ([Ca2+]i) in smooth muscle cells. We previously demonstrated that BKCa channels play an important role in regulating SO motility. At the early stage of SOD, BKCa channel is insensitive to the increase of [Ca2+]i in SO smooth muscle cells and the activity of BKCa channel is down-regulated. However, its underlying mechanism is not clear. The alternative splicing of STRess axis-regulated Exon (STREX) can regulate the activity of the BKCa channel through changing the susceptibility of phosphorylation modification of BKCa channel α-subunit, which ultimately affects the cell function. To confirm the hypothesis that the decreased alternative splicing of STREX plays important role for the tyrosine phosphorylation modification of BKCa channel α-subunit, the decrease of channel activity as well as the impaired SO motility, we plan to combine the molecular biology, patch clamp and confocal technique to reveal the molecular mechanism for alternative splicing of STREX that regulate the BKCa channel activity via controlling tyrosine phosphorylation modification of BKCa channel α-subunit. This study will contribute to clarifying the underlying mechanism of SOD, as well as providing a novel theoretical basis for the identification and prevention of SOD.

Oddi括约肌功能紊乱（SOD）易引发肝、胆和胰功能紊乱，明确其发病机制具有重要的临床意义。Oddi约肌（SO）舒缩功能异常是引发SOD的关键原因，其可能机制是大电导钙激活钾离子通道（BKCa）抑制平滑肌细胞 [Ca2+]i浓度超载的调控机制异常。课题组前期研究表明BKCa通道调节SO的运动功能，在SOD前期，SO细胞BKCa通道对升高的[Ca2+]i失敏，通道活性下调，但其分子机制不明。应激外显子（STREX）的选择性剪接可改变BKCa通道α-亚基磷酸化易感性，调节通道活性，进而影响细胞功能。本课题围绕“STREX选择性剪接减弱是SO细胞BKCa通道α-亚基酪氨酸磷酸化修饰异常、通道活性下降及SO运动功能损伤的关键调控机制”这一科学假说，拟综合分子生物学、膜片钳和共聚焦等技术，探索STREX选择性剪接调控α-亚基磷酸化，改变BKCa活性的分子机制，阐明SOD发生机理，为SOD的诊断和防治提供理论基础。

本课题是针对高胆固醇饮食损伤Oddi括约肌（SO）运动性功能的机制研究，初步阐明了可变外显子STREX调控BKCa通道在高胆固醇血症（HC）导致的SO功能紊乱（SOD）中的作用机制。课题以STREX外显子调控BKCa通道α-亚基酪氨酸磷酸化修饰水平及通道Ca2+敏感性为切入点，采用免疫荧光、免疫沉淀、蛋白印迹、荧光实时定量PCR、病毒感染、激光共聚焦、电生理、肌环张力检测及药物干预等方法探讨STREX选择性剪接介导BKCa通道活性调控兔SO运动功能的分子机制。研究结果显示：建立HC模型能很好的模拟SOD形成的早期过程，构建HC兔SO平滑肌细胞（RSOSMCs）模型也能较好的模拟SOD形成过程中的体外细胞模型；连续高胆固醇饮食8-12周可严重损伤兔SO运动性，5 μM胆固醇溶液干预细胞24h可显著干扰RSOSMCs的兴奋性变化；HC条件下，STREX选择性接入减少，RSOSMCs [Ca2+]i超载，BKCa通道α-亚基酪氨酸磷酸化修饰水平下降，通道活性显著降低，模型兔SO离体肌环张力显著升高，导致兔SO运动功能损伤；而HC诱导STREX的选择性接入减少，使得BKCa通道对Ca2+的敏感性降低，从而下调通道α-亚基酪氨酸磷酸化易感性，降低通道活性，最终增加SO细胞异常的兴奋性收缩，引发SOD，这可能是高胆固醇饮食引起的SOD形成过程中，STREX可变外显子调控兔SO细胞BKCa通道活性进而控制兔SO运动性的分子作用机制；且抑制Src可显著降低STREX的选择性接入，从而下调BKCa通道活性和SO运动性，而抑制CaMK IV会产生与抑制Src相反的作用；因此，Src和CaMK IV极可能分别是兔SO细胞STREX接入的关键激活因子和抑制因子。本课题初步明确了STREX选择性剪接控制通道α-亚基酪氨酸磷酸化修饰，调节通道活性的作用机制，有望为阐明高胆固醇饮食诱发SOD乃至胆结石的分子机制提供新的研究思路和实验基础，同时也为胆固醇胆结石的防治提供了新的干预靶点。