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

摘要 摘要肺动脉高压(PAH)是一种少见的慢性人类疾病，具有较高的发病率和死亡率。 费率。目前的治疗方法在改善PAH的生活质量和延长寿命方面的益处非常有限 病人。编码钙激活氯离子的基因TMEM16A或ANO1的表达 肺动脉平滑肌细胞(PASMC)的钙通道(CaCC)增强 肺动脉高压(PH)。ANO1编码的CaCC被认为是一种兴奋机制 对血管收缩药如5-羟色胺的反应。基于从MICE收集的新的初步数据 仅在平滑肌细胞中表达一种名为GCaMP3的钙离子生物传感器蛋白，推测的机制是 将血管收缩活性与ANO1联系起来的兴奋-收缩偶联(EC偶联)需要 根据5-羟色胺产生的非常稳定的肺动脉收缩依赖于 ANO1介导的膜高度局域周期性肌质网钙释放和再摄取之间的微妙平衡 去极化和钙离子通过CaV1.2通道进入。EC偶联是如何改变的，增强了EC的作用 ANO1在PH中的表达和功能尚不清楚。我们最近报道，肺组织中CaCCs的活性 动脉平滑肌细胞受磷脂酰肌醇4，5二磷酸(PIP2)直接结合的抑制 膜磷脂调节许多膜蛋白，包括离子通道和转运蛋白。这 发现意义重大，尽管存在争议，因为在这一新范式的背景下，ANO1被激活 将由受体刺激后的双重自我强化机制在生理上触发 导致肺循环血管收缩。本申请中的初步数据显示，ANO1 在PH的慢性低氧小鼠模型中，调节PIP2水平的几种关键酶水平升高。这个 控制PIP2水平的酶的结构排列和ANO1调节，这一点的影响 与PIP2相互作用的ANO1结构域(S)与肺动脉张力的关系尚不清楚。vbl.使用 多学科方法和几种复杂的转基因条件性ANO1基因敲除动物模型 以及小鼠和人的PASMC，我们将检验这一假设，即通过 ANO1通道PIP2改变在慢性PASMCs功能重构中起关键作用 低氧诱导的PH。提出了三个具体目标来检验这一假设：具体目标1：确定 ANO1通道在血管收缩和激动剂引起的PA局部钙振荡中的作用 来自正常小鼠和PH小鼠。具体目标2：该委员会的结构组织和职能意义是什么 PH？ANO1通道微区与PIP2代谢的调节具体目标3：什么是 PIP2对心肌细胞天然ANO1通道调控的生物物理和分子机制 PASMC和HEK-293细胞过表达ANO1？

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?