Nongenomic bile acid on smooth muscle BK channels

平滑肌 BK 通道上的非基因组胆汁酸

• 批准号: 6891407
• 负责人: ALEX M. DOPICO
• 金额: $ 18.25万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891407
• 关键词: Xenopus; Xenopus oocyte; blood vessel disorder; chemical structure function; chimeric proteins; cholanate compound; cholesterol; intermolecular interaction; laboratory rat; molecular biology; muscle cells; muscle relaxation; pathologic process; pharmacology; posttranslational modifications; potassium channel; protein engineering; protein structure; vascular smooth muscle; vasodilation; vasodilators; voltage /patch clamp

DESCRIPTION (provided by applicant): Previous data from our laboratory demonstrated that acute exposure to cholane derivatives (bile acids and synthetic analogs) increases the activity of large conductance, Ca2+-sensitive K+ (BK) channels in arterial smooth muscle. Activation of smooth muscle BK channels is thought to be a major mechanism underlying vasodilation caused by not only bile acid derivatives, but also other physiologically relevant steroids, such as estradiol, progesterone, and androgens. In contrast, others and we found that increased cholesterol levels lead to smooth muscle BK channel inhibition. The long-term goal of this proposal is to identify both the structural determinants in the steroid molecule and the channel protein regions that are responsible for steroid activation of BK channels. Combining methodologies from Electrophysiology (patch-clamp), Molecular Biology, and Pharmacology, we will specifically address: 1) which chemical determinants in the cholane derivative molecule are critical for activating smooth muscle BK channels; 2) which channel subunit (alpha vs. beta) is necessary/sufficient for cholane derivative action; 3) which protein regions in the channel subunits are responsible for conferring cholane sensitivity to the ion channel complex; 4) whether modulation of BK channel activity by cholane derivatives underlies smooth muscle relaxation caused by these steroids. We will answer these questions going from studies in isolated arteries to evaluate drug action on tissue physiology to studies that used engineered ion channel subunits expressed in Xenopus oocytes to address the more mechanistic aspects of the application. We will initially use the rat cerebral artery to freshly isolate both a resistive artery segment to measure changes in tone caused by steroids, and dissociated myocytes to probe steroid modulation of native BK channels. The same overall strategy can be applied to other relevant vessels in the future. We chose the rat cerebral artery/myocyte model because: a) the key role of BK channel activity as regulator of arterial tone was clearly demonstrated; b) we have experience studying the effect of modulators on the active/passive tone of isolated rat cerebral arteries; c) we have isolated, cloned and successfully expressed in Xenopus oocytes the pore forming subunit of the rat cerebral artery BK channel; d) the therapeutic need for new cerebral vasodilators that act on the smooth muscle itself, i.e., that can be effective in the absence of intact endothelial function. Pinpointing the molecular mechanisms involved in the steroid-cerebrovascular BK channel interaction will bring fundamental insight for the rationale design of steroidal vasodilators of potential therapeutic use, devoid of hormonal side effects.

描述（由申请方提供）：我们实验室的既往数据表明，急性暴露于胆烷衍生物（胆汁酸和合成类似物）可增加动脉平滑肌中大电导、Ca 2+敏感性K+（BK）通道的活性。平滑肌BK通道的激活被认为是血管舒张的主要机制，不仅由胆汁酸衍生物引起，还由其他生理学相关的类固醇如雌二醇、孕酮和雄激素引起。相反，其他人和我们发现，胆固醇水平增加导致平滑肌BK通道抑制。该提案的长期目标是确定类固醇分子中的结构决定因素和负责类固醇激活BK通道的通道蛋白区域。结合电生理学的方法（膜片钳）、分子生物学和药理学，我们将具体讨论：1）胆烷衍生物分子中的哪些化学决定簇对于激活平滑肌BK通道是关键的; 2）哪个通道亚基（α对β）对于胆烷衍生物作用是必要/充分的; 3）通道亚基中的哪些蛋白质区域负责赋予胆烷对离子通道复合物的敏感性; 4）胆烷衍生物对BK通道活性的调节是否是这些类固醇引起的平滑肌松弛的基础。我们将回答这些问题，从孤立的动脉研究，以评估药物对组织生理学的作用，研究使用工程化的离子通道亚基在非洲爪蟾卵母细胞中表达，以解决更多的机械方面的应用。我们将首先使用大鼠脑动脉新鲜分离的阻力动脉段，以测量由类固醇引起的张力变化，和解离的肌细胞，以探测天然BK通道的类固醇调制。同样的总体战略可以在未来应用于其他相关船舶。我们选择大鼠脑动脉/肌细胞模型是因为：a）BK通道活性作为动脉张力调节剂的关键作用已被清楚地证明; B）我们有研究调节剂对分离的大鼠脑动脉的主动/被动张力的作用的经验; c）我们已经分离、克隆并在爪蟾卵母细胞中成功表达了大鼠脑动脉BK通道的孔形成亚基; d）对作用于平滑肌本身的新的脑血管扩张剂的治疗需求，即，在缺乏完整内皮功能的情况下有效。精确定位类固醇脑血管BK通道相互作用的分子机制将为潜在治疗用途的类固醇血管扩张剂的合理设计带来基本的见解，没有激素副作用。