Fox蛋白在血管平滑肌CaV1.2钙离子通道剪接体的调控中的作用及其在高血压中的应用

L-type CaV1.2 channels have a dominant role in arterial smooth muscle contraction and its dysregulation is therefore important in hypertension. Splicing events, as an important mechanism of post-transcript modulation in L-type calcium channels, has been found to invovle in the neural development, and which is regulated by Fox proteins. Whether Fox proteins take part in the modulation of CaV1.2 calcium channel splicing in the hypertensive arteries remains unknown. Our preliminary data indicated the expressiones of the alternative splice variants of CaV1.2 calcium channels have been changed: the expression of exon 9* increased; while exon 33 decreased. More importantly, the expression of the Fox proteins also changed in the hypertensive arteries, which was associated with the level of exon 9* and exon 33. Therefore, we presume that Fox proteins regulate the splicing of CaV1.2 calcium channels in the arteries, in turn change these channel properties, thus modulate the arterial constriction and blood pressure. Here, we will use molecular biological techniques, whole-cell patch clamp, in-vitro arterial constriction assay and in-vivo blood pressure monitor to investigate the modulation of CaV1.2 calcium channel splicing by Fox proteins from cellualr, organ and even whole animal levels. This project can illustrate an important mechanism, which never known before, in the development of hypertension, and may provide a newly promissing theraputic target in the management of vascular hypertension.

L型钙通道CaV1.2在动脉平滑肌收缩中起着非常重要的作用，其功能紊乱与高血压密切相关。剪接体作为调控钙离子通道一个重要的转录后调控机制，已发现在神经发育中受到Fox蛋白的调控。但Fox蛋白是否参与高血压血管中L型钙离子通道的剪接体调控目前还不清楚。我们的前期研究表明高血压大鼠血管内CaV1.2剪接体exon 9*表达升高，而exon 33表达降低。更为重要的是，血管内Fox蛋白的表达也降低。所以，我们推测Fox蛋白调控血管中CaV1.2剪接体表达改变，从而改变血管平滑肌钙离子通道特征，进而调控血管舒缩和血压。在此，我们将利用过表达质粒、siRNA和转录扫描等分子生物学技术，并利用全细胞膜片钳、体外血管舒缩检测和在体血压检测等生理学实验在细胞、器官和整体水平验证Fox蛋白对CaV1.2剪接体的调控作用。这一项目将为我们提供高血压发病的一个重要机制，并有望为治疗高血压提供一个崭新的治疗靶点。

在血管平滑肌细胞中，通过CaV1.2电压依赖型钙离子通道的钙内流对于维持血管肌源性张力和血压起着至关重要的作用；而譬如选择性剪接的转录后调控机制可以优化CaV1.2钙离子通道的功能。目前已经知道剪接调控因子Rbfox在神经发育过程中调控了CaV1.2 pre-mRNA剪接事件，但Rbfox在血管CaV1.2功能调节和血管张力中的作用还不清楚。通过Real-time RT-PCR和Western blotting，我们发现Rbfox2比它的同源体Rbfox1在血管平滑肌表达更加丰富，提示Rbfox2在血管中将起着主要作用。相较于正常血管，我们发现高血压血管中CaV1.2可变剪接外显子exon 9*增加10.3%，而外显子exon 33下降约10.5%；同时，未预料到的是Rbfox2的表达增加约3倍。在血管平滑肌中，siRNA介导的Rbfox2敲降能动态调控CaV1.2可变剪接，其exon 9*明显升高，而exon 33明显下降。利用膜片钳技术，我们发现Rbfox2降低所导致的CaV1.2异常剪接导致CaV1.2钙通道的稳态激活和失活曲线向超极化方向移动，这使得钙窗电流电位变得更负。而且，敲降Rbfox2导致大鼠肠系膜动脉压力诱导的肌源性张力增高。总之，我们的数据说明了Rbfox2可以通过动态调控CaV1.2可变剪接外显子exon 9*和33来调节血管CaV1.2钙通道的功能，从而影响血管肌源性张力。这些提示Rbfox2在血管性高血压中起着非常重要的作用。