雌二醇经雌激素受体核启动信号通路抑制肌细胞Kir电流的机制研究

Inwardly rectification potassium channel 2.1 (Kir2.1) is a crucial transmembrane protein mainly stabilizing rest membrane potential in cardiomyotes, skeletal muscle cells and neurons. The mutations in the encoding gene KCNJ2 are responsible for Andersen-Tawil syndrome (ATS) with a clinical triad comprised of periodic paralysis, cardiac arrhythmia and developmental dysmorphia. The cardiac disturbances in ATS patients tend to be ventricular arrhythmia and even lead to sudden death, thus it is the most lethal muscle ion channel disease in juveniles and there is no effective treatment. We noticed that serum estrogen elevation may exacerbate the cardiac and skeletal muscle involvement during a follow-up of 18 Chinese ATS patients. To further investigate the mechanism underlying estrogen-dependent modulation of Kir2.1, we recorded the kir currents via whole cell patch clamp in HEK293T cells overexpressed KCNJ2 WT before or after 17β-estradiol incubation. The current density kept unchanged with acute perfusion, but it was significantly reduced after long-term incubation. Analysis of mRNA with RT-PCR technique revealed that KCNJ2 gene was repressed by 17β-estradiol. Taken together, estrogen repressed kir2.1 through downregulation in gene transcription by nuclear-initiated estrogen receptor signaling pathway. Promisingly, the implementation of current program would substantially deepen our understanding of the underlying mechanism of the modulation of Kir2.1 in muscle cells, and potentially provide novel therapeutic strategy for skeletal ion channel disease and cardiac arrhythmia.

内向整流钾离子通道2.1（Kir2.1）是广泛分布于心肌、骨骼肌及神经元上维持细胞膜电位稳定的重要蛋白，其责任基因KCNJ2突变后将引起具备周期性麻痹、心律失常及外形异常三主征的Andersen-Tawil综合征（ATS），迄今为止无明确的治疗手段。在我们前期的临床观察中，发现雌激素可能加重心脏及骨骼肌的受累。进而在体外给予过表达KCNJ2的细胞雌二醇孵育见Kir电流降低，从而印证了临床的观察结果。KCNJ2的mRNA水平在雌二醇干预后显著下降，提示这一作用主要经核启动的雌激素信号通路影响转录而实现。本研究旨在通过确定KCNJ2是否为雌二醇/雌激素受体复合物作用的靶基因，探索转录因子Sp1及Sp3的协同调控作用，将有助于加深对雌激素抑制肌细胞Kir电流机制的理解，从而为骨骼肌离子通道病和心律失常探索新的治疗靶点。

内向整流钾离子通道2.1（Kir2.1）是负责维持心肌、骨骼肌及神经元上细胞膜电位稳定的重要蛋白，其责任基因KCNJ2突变后将引起Andersen-Tawil综合征（ATS）。临床ATS队列观察发现女性患者心律失常严重、周期性麻痹发作频繁，一对ATS母女的纵向随访数据亦证实心脏和骨骼肌受累在青春期尤为显著。我们进而在体外给过表达KCNJ2的细胞予雌二醇孵育，膜片钳技术检测可见Kir电流显著降低并呈浓度依赖性，从而印证了临床的观察结果。进一步探索雌二醇抑制Kir电流的机制，我们发现雌二醇及受体复合体下游的协同转录因子Sp蛋白家族（Sp1及Sp3）的mRNA表达量亦显著升高，提示雌二醇对Kir电流的抑制很可能是通过转录调控而实现。我们对雌二醇复合体-Sp蛋白家族转录调控靶基因进行检测，发现PI3K-1A/1B/CA/CB的mRNA以及PI3K p85蛋白表达量显著升高。PI3K的激酶下游催化底物PIP2为Kir2.1离子通道发挥正常作用的重要协同分子，因此雌二醇经核转导通路上调PI3K表达从而抑制Kir电流，可能是雌二醇加重ATS的重要病理机制，而雌二醇复合体-Sp蛋白家族及PI3K激酶轴则可为ATS青春期心律失常的一个潜在治疗靶点。