Role of ANO1 Channels and Its Regulation by PIP2 in EC-Coupling in Pulmonary Artery Myocytes

总结 肺动脉高压（PAH）是一种罕见的慢性疾病，具有较高的发病率和死亡率 rates.目前的治疗在PAH的生活质量改善和寿命方面提供的获益非常有限 患者TMEM 16 A或Anoctamin 1（ANO 1）基因的表达，该基因编码Ca 2+激活的Cl- 肺动脉平滑肌细胞（PASMCs）中钙通道（CaCCs）的表达增强。 肺动脉高压（PH）。ANO 1编码的CaCC被认为是一种兴奋机制， 对血管收缩剂如血清素的反应。根据从老鼠身上收集到的新的初步数据， 仅在平滑肌细胞中表达称为GCaMP 3的Ca 2+生物传感器蛋白， 兴奋-收缩偶联（EC偶联）将血管收缩剂的活性与ANO 1联系起来， 重新审视的基础上，非常稳定的收缩肺动脉产生的血清素依赖于一个 高度局部化的周期性SR Ca 2+释放和再摄取之间的精细平衡，ANO 1介导的膜 去极化和Ca 2+通过CaV1.2通道进入。EC偶联是如何改变的， ANO 1在PH中的表达和功能尚不清楚。我们最近报道了肺组织中CaCCs的活性， 动脉平滑肌细胞被磷脂酰肌醇4，5二磷酸（PIP 2）的直接结合所抑制， 膜磷脂调节许多膜蛋白，包括离子通道和转运蛋白。这 这一发现意义重大，尽管存在争议，因为在这种新的范式背景下，ANO 1的激活 在刺激受体后， 导致肺循环中的血管收缩。本申请中的初步数据显示， 在PH的慢性缺氧小鼠模型中，调节PIP 2水平的几种关键酶升高。 控制PIP 2水平和ANO 1调节的酶的结构排列， 与肺动脉张力和与PIP 2相互作用的ANO 1结构域的关系尚不清楚。使用 多学科方法和几种复杂的转基因条件性ANO 1敲除动物模型 以及小鼠和人PASMCs，我们将检验以下假设： 慢性肺动脉高压大鼠肺动脉平滑肌细胞ANO 1通道的PIP 2改变并在其功能重建中起关键作用 提出了三个具体目标来检验这一假设：具体目标1：确定 ANO 1通道在PA激动剂引起的血管收缩和局部Ca 2+振荡中的作用 正常和PH小鼠。具体目标2：组织的结构和功能意义是什么？ ANO 1通道微区和PIP 2代谢在PH？具体目标3： 参与PIP 2调节天然ANO 1通道的生物物理和分子机制， PASMCs和HEK-293细胞过表达ANO 1？

Summary Pulmonary arterial hypertension (PAH) is a rare chronic human disease exhibiting high morbidity and mortality rates. Current treatments offer very limited benefits in terms of quality of life improvement and longevity to PAH patients. The expression of the gene TMEM16A or Anoctamin1 (ANO1) that encodes for Ca2+-activated Cl- channels (CaCCs) is enhanced in pulmonary artery smooth muscle cells (PASMCs) from animal models of pulmonary hypertension (PH). ANO1-encoded CaCCs are believed to act as an excitatory mechanism in response to vasoconstrictors such as serotonin. Based on new preliminary data gathered from mice expressing a Ca2+ biosensor protein called GCaMP3 only in smooth muscle cells, the postulated mechanism of excitation-contraction coupling (EC-coupling) linking the activity of vasoconstrictors to ANO1 needs to be revisited on the basis that the very stable contraction of pulmonary arteries produced by serotonin relies on a fine balance between highly localized cyclical SR Ca2+ release and reuptake, ANO1-mediated membrane depolarization and Ca2+ entry through CaV1.2 channels. How EC-coupling is altered and the role of increased ANO1 expression and function in PH is unknown. We recently reported that the activity of CaCCs in pulmonary artery smooth muscle cells is inhibited by direct binding of phosphatidylinositol 4,5 bisphosphate (PIP2), a key membrane phospholipid regulating many membrane proteins including ion channels and transporters. This discovery is significant, albeit controversial, because in the context of this new paradigm activation of ANO1 would be physiologically triggered by a dual self-reinforcing mechanism following stimulation of receptors leading to vasoconstriction in the pulmonary circulation. Preliminary data in this application show that ANO1 and several key enzymes regulating PIP2 levels are elevated in the chronic hypoxic mouse model of PH. The structural arrangement of the enzymes controlling PIP2 levels and ANO1 regulation, the impact of this relationship on pulmonary arterial tone and the domain(s) of ANO1 that interact with PIP2 are unknown. Using a multidisciplinary approach and several sophisticated transgenic conditional ANO1 knockout animal models and mouse and human PASMCs, we will test the hypothesis that the expression, function and regulation by PIP2 of ANO1 channels are altered and play a key role in the functional remodeling of PASMCs in chronic hypoxia-induced PH. Three specific aims are proposed to test this hypothesis: Specific Aim 1: To determine the role of ANO1 channels in the vasoconstriction and localized Ca2+ oscillations elicited by agonists in the PA from normal and PH mice. Specific Aim 2: What is the structural organization and functional significance of the ANO1 channel microdomain and regulation by PIP2 metabolism in PH? Specific Aim 3: What are the biophysical and molecular mechanisms involved in the modulation by PIP2 of native ANO1 channels in PASMCs and HEK-293 cells over-expressing ANO1?