STREX剪切变异体在结肠平滑肌BK通道的非钙依赖调控中的作用及机制研究

Large-conductance calcium-activated potassium channel (BK) is the key potassium channel that affects the smooth muscle electro-mechanical activities. The regulation mechanisms of BK and its effect on gastrointestinal smooth muscle motility are lack of intensive research which limits the application of related strategy on ion channel in diagnosis and treatment of gastrointestinal (GI) motility disease. Our previous studies found that BK was highly expressed in the distal colon smooth muscle. Besides the classical intracellular calcium level dependent regulation, BK was also activated by calcium-independent regulatory pathway, the mechanism is unclear and the role of STREX splice variants in α-subunit can’t be ignore. In the current project, the expression patterns of BK in colonic smooth muscle strips, freshly isolated colonic smooth muscle cells and cell lines overexpressed/knockdown STREX will be detected. Tension of isolated perfused colonic smooth muscle strips will be measured to explore the role of BK in tension regulation under basic, stretched and cholinergic excited conditions and the relevance of STREX splice variants will be analyzed. Calcium sensitivity, voltage sensitivity and mechanosensitivity of BK will be investigated by combined use of patch clamp and calcium imaging techniques. Current injection method will be used to simulate slow wave potentials to induce action potentials and intracellular calcium levels will be clamped to study calcium-independent regulation of BK with different phenotypes under basic and cholinergic excited conditions. The related molecular mechanism will be explored by intervening related signal. This study will ascertain that STREX splice variant is the key structure invovled in calcium-independent regulation of BK in colonic smooth muscle, which will provide a new theoretical foundation for the study of the ion channel mechanism of colon tension regulation and provide new clues for the study of pathogenesis and treatment strategy of GI motility disorder diseases.

BK通道是影响平滑肌电-机械活动的关键钾通道，其对胃肠平滑肌动力的影响及自身调控机制缺少深入研究，制约了胃肠动力紊乱疾病的诊疗。我们前期研究发现BK在远端结肠平滑肌高表达，除受经典的胞内钙水平调控外，还存在非钙依赖调控途径，但机制不清，α亚基STREX剪切变异体的作用不容忽视。本课题拟检测不同节段结肠平滑肌肌条、急性分离肌细胞和过表达/干扰STREX的细胞系中BK的表达类型、水平和定位；利用离体灌流结肠平滑肌条研究BK对张力的影响及与STREX剪切变异体表达水平的关系；联合应用膜片钳和钙成像技术检测BK的钙、电压和机械敏感性；电流注入法模拟慢波电位和诱导动作电位，钳制胞内钙水平，研究基础状态下和胆碱激动时不同表型BK非钙依赖调控特点，并干预相关信号分子探讨机制。本课题将阐明STREX剪切变异体是结肠平滑肌BK非钙依赖调控的关键结构，为揭示结肠张力调节的离子通道机制提供新的理论依据。

BK通道是影响平滑肌电-机械活动的关键钾通道，其对胃肠平滑肌动力的影响及自身调控机制缺少深入研究，制约了胃肠动力紊乱疾病的诊疗。我们前期研究发现BK在远端结肠平滑肌高表达，除受经典的胞内钙水平调控外，还存在非钙依赖调控途径，但机制不清，α亚基STREX剪切变异体的作用不容忽视。本课题检测了不同节段结肠平滑肌肌条、急性分离肌细胞和过表达STREX的细胞系中BK的表达类型、水平和定位；利用离体灌流结肠平滑肌条研究BK对张力的影响及与STREX剪切变异体表达水平的关系；应用膜片钳和压力钳技术检测BK的机械敏感性；研究基础状态下和胆碱激动时不同表型BK非钙依赖调控特点。研究结果发现，远端结肠平滑肌中BK通道的表达高于消化道其它位置；胆碱能受体激活后会通过M3受体激活下游PKC，进而抑制ZERO型BK通道，使结肠平滑肌肌张力进一步增强，但PKC对STREX型BK通道无明显影响，可能为胃肠平滑肌动力调节机制的研究提供新的方向。进一步研究发现，β1亚基胞外环可以感受机械刺激并激活BK通道，是独立于STREX序列来调节BK通道牵张敏感性的重要机制。此外，BK通道参与了胰岛素信号通路的调节。胰岛素/PI3K可以非钙依赖的激活STREX型BK通道。本课题揭示了BK通道非钙依赖性调控的机制，并探究了其在结肠平滑肌张力调节过程中的作用，为研究离子通道的生理功能和调控机制提供了新的思路，可能为胃肠动力紊乱等疾病的发病机制和治疗策略提供新线索。