Vascular control by K+ channel trafficking

描述(申请人提供)：小(阻力大小)大脑动脉和小动脉控制局部脑血流，从而为神经元和其他脑细胞提供必要的氧气和营养。动脉收缩的一个重要调节因素是由离子通道控制的平滑肌细胞膜电位。高血压与中风和痴呆症等脑部疾病的风险增加有关。高血压患者的大脑动脉被去极化，导致收缩能力增强，但参与这种病理变化的机制尚不清楚。以往的研究主要集中在识别调节位于动脉平滑肌细胞质膜上的离子通道活性的机制。相比之下，控制平滑肌细胞质膜上功能性离子通道及其辅助亚单位数量的机制尚不清楚。大电导钙(Ca~(2+))-激活钾(BKCa)通道是动脉平滑肌细胞膜电位和收缩能力的主要生理调节剂。动脉平滑肌细胞表达两个BKCa通道亚基：一个是孔道形成亚基(BK)，另一个是通道生理功能所必需的辅助亚基。这一应用源于新的初步数据表明，生理刺激控制BK和1的表面表达，以调节通道亚单位组成、通道活动和动脉收缩。我们还提供了证据表明，高血压与调节BKCa通道亚单位表面表达从而促进血管收缩的机制的病理变化有关。三个特定的目标将被用来检验中央假说，即血管调节刺激调节BKCa通道亚单位的表面表达以控制脑动脉收缩，以及这些过程的病理改变导致与脑血管疾病相关的血管收缩。目的1验证血管扩张剂和缩血管药调节1亚单位表面表达以控制平滑肌细胞BKCa通道活性和动脉收缩的假说。目的2探讨血管紧张剂和血管扩张剂调节BK？表面表达和降解，从而调节平滑肌细胞BKCa通道活性和动脉收缩的假说。目的3将探讨高血压与刺激血管收缩的BK？和？1亚单位表面表达的功能障碍有关的假说。用于验证这些假说的方法包括动脉生物素化、FRET、RNAi、co-IP、免疫荧光、膜片钳电生理、膜电位记录、细胞内钙成像和动脉肌成像。这一建议将为通过控制BKCa通道表面表达的生理和病理机制来调节脑动脉收缩性能提供重要的新信息。

DESCRIPTION (provided by applicant): Small (resistance-size) cerebral arteries and arterioles control regional brain blood flow, thereby providing neurons and other brain cells with necessary oxygen and nutrients. An essential regulator of artery contractility is smooth muscle cell membrane potential, which is controlled by ion channels. Hypertension is associated with increased risk for cerebral diseases, including stroke and dementia. Cerebral arteries from hypertensive subjects are depolarized, leading to elevated contractility, but mechanisms involved in this pathological alteration are unclear. Previous studies have focused on identifying mechanisms that regulate the activity of ion channels located in the plasma membrane of arterial smooth muscle cells. In contrast, mechanisms that control the number of functional ion channels and their auxiliary subunits in the plasma membrane of smooth muscle cells are unclear. Large-conductance calcium (Ca2+)-activated potassium (BKCa) channels are a major physiological modulator of arterial smooth muscle cell membrane potential and contractility. Arterial smooth muscle cells express two BKCa channel subunits: a pore-forming ¿(BK¿) and an auxiliary ¿1 that is essential for channel physiological functions. This application stems from novel preliminary data indicating that physiological stimuli control BK¿ and ¿1 surface expression to modulate channel subunit composition, channel activity and arterial contractility. We also provide evidence that hypertension is associated with pathological alterations in mechanisms that regulate BKCa channel subunit surface expression, thereby promoting vasoconstriction. Three specific aims will be investigated to test the central hypothesis that vasoregulatory stimuli modulate surface expression of BKCa channel subunits to control cerebral artery contractility, and that pathological modification of these processes leads to vasoconstriction associated with cerebrovascular disease. Aim 1 will examine the hypothesis that vasodilators and vasoconstrictors regulate ¿1 subunit surface expression to control BKCa channel activity in smooth muscle cells and arterial contractility. Aim 2 will investigate the hypothesis that vasoconstrictors and vasodilators modulate BK¿ surface expression and degradation to regulate smooth muscle cell BKCa channel activity and arterial contractility. Aim 3 will explore the hypothesis that hypertension is associated with dysfunctional control of BK¿ and ¿1 subunit surface expression that stimulates vasoconstriction. Methods used to test these hypotheses will include arterial biotinylation, FRET, RNAi, co-IP, immunofluorescence, patch-clamp electrophysiology, membrane potential recording, intracellular Ca2+ imaging and arterial myography. This proposal will provide significant novel information concerning cerebral artery contractility regulation by physiological and pathological mechanisms that control BKCa channel surface expression.