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(申请人提供)：我们实验室以前的数据表明，急性暴露于胆烷衍生物(胆汁酸和合成类似物)会增加动脉平滑肌上大电导、钙敏感的K+(BK)通道的活性。平滑肌BK通道的激活不仅被认为是胆酸衍生物引起的血管扩张的主要机制，也被认为是其他与生理相关的类固醇如雌二醇、孕酮和雄激素引起的主要机制。相反，其他人和我们发现，胆固醇水平的增加会导致平滑肌BK通道的抑制。这项建议的长期目标是确定类固醇分子和通道蛋白区域中负责类固醇激活BK通道的结构决定因素。结合电生理学(膜片钳)、分子生物学和药理学的方法，我们将具体讨论：1)胆烷衍生物分子中的哪些化学决定因素是激活平滑肌BK通道的关键；2)哪个通道亚单位(α和β)是胆烷衍生物作用的必要/充分条件；3)通道亚单位中的哪个蛋白质区域负责赋予离子通道复合体对胆碱的敏感性；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，从评估药物对组织生理学作用的离体动脉研究，到使用非洲爪哇卵母细胞表达的工程离子通道亚单位来解决应用的更多机械方面的研究。我们最初将使用大鼠大脑动脉新鲜分离阻力动脉段，以测量类固醇引起的张力变化，并分离心肌细胞，以探索类固醇对天然BK通道的调节。未来，同样的总体战略也可以应用于其他相关船只。我们之所以选择大鼠大脑动脉/心肌细胞模型，是因为：a)BK通道活性作为动脉张力调节器的关键作用已经得到明确的证实；b)我们有研究调节剂对大鼠离体脑动脉主动/被动张力的影响的经验；c)我们已经分离、克隆并成功地在非洲爪哇卵母细胞中表达了大鼠大脑动脉BK通道的成孔亚单位；d)治疗方面需要新的作用于平滑肌本身的脑血管扩张剂，即在缺乏完整的内皮功能的情况下可以有效地发挥作用。明确类固醇-脑血管BK通道相互作用的分子机制将为设计无激素副作用的类固醇血管扩张剂提供基本的理论依据。