REGULATION OF K CHANNELS IN CORONARY SMOOTH MUSCLE

本提案的长期目标是定义生理学 钙激活钾通道(Kca)和三磷酸腺苷敏感钾通道(KATP)的作用 冠状动脉平滑肌，通过研究它们的生物物理特性， 它们对血管活性物质的反应及其调节机制(S)。 主要假说假设K+通道可能受 血管活性物质和细胞内信使。因此，他们的活动 可以控制静息电位，从而控制冠脉张力。 两种主要类型的K+通道在血管中具有特征 肌肉、钙激活(KCA)和ATP敏感的K+(KATP)通道。 然而，人们对细菌的多样性和监管特性知之甚少 这些通道位于冠脉平滑肌(CSM)。我们的初步调查结果 提示CSM具有KCA和KATP通道。KCA频道有 丰富，具有不同的功能特性，并可能被抑制 血管收缩药(血管紧张素II、血栓素)的外用 A2、TXA2)，并由内部应用的G蛋白(Galphas)激活。因此， 我计划解决的关键问题是：什么是生物物理和 CSM中KCA和KATP通道的药理特性？是 这些通道在静息细胞中自发活跃吗？什么是 三磷酸腺苷抑制KATP通道的机制？其作用机制是什么？ 血管活性物质对KCA通道的作用，G蛋白是否参与？ KATP通道是否也受血管活性物质的调节？ 为了回答这些问题，我计划：1)进一步研究生物物理学和 KCA和KATP通道的药理特性；2) 研究AgII和TXA2对KCA通道的作用，并探讨其作用机制 这些血管活性物质对KATP通道的潜在作用；3) 确定AGII和TXA2对KCA通道的调制机制(S) (直接配基门控，直接G蛋白门控“膜分隔”，和G 蛋白质介导的代谢途径导致磷酸化和/或 将探索花生四烯酸(AA)代谢)；4)研究是否 去磷酸化修饰AGII和TXA2的作用， 以及5)调查KCA和/或KATP通道是否受 血管松弛药，如AA和前列腺素I2，以及它们的作用方式。 为了达到拟议的目标，我将采用综合方法，即 包括使用：a)膜片钳技术(全细胞、细胞 附加的和无细胞的贴片)在单个细胞上，以及b)通道 在功能上重组成双分子层。要进行膜片钳研究，需要 已经开发出一种从冠脉肌肉中提取的单细胞制剂。在……里面 此外，我还成功地分离出了一种浓缩的冠脉质膜 允许在双分子层Kca和KCa中官能化重建的部分 KATP通道。 这些研究将有助于理解冠状动脉张力是如何调节的， K+通道开放剂如何缓解心血管疾病，以及 可能是为了设计减少冠状动脉痉挛的治疗方法 对心肌缺血和心脏功能障碍负责。

The long term goal of the present proposal is to define the physiological role of calcium-activated (KCa) and ATP-sensitive (KATP) K channels from coronary smooth muscle, by studying their biophysical characteristics, their response to vasoactive substances and their regulatory mechanism(s). The main hypothesis postulates that K+ channels may be modulated by vasoactive substances and intracellular messengers. Thus, their activity can control the resting potential and consequently the coronary tone. Two major types of K+ channels have been characterized in vascular smooth muscle, Ca2+-activated (KCa) and ATP-sensitive K+ (KATP) channels. However, little is known about the diversity and regulatory properties of these channels in coronary smooth muscle (CSM). Our initial findings indicate that CSM possesses KCa and KATP channels. KCa channels are abundant, have different functional properties, and may be inhibited by external application of vasoconstrictors (angiotensin II, AgII; thromboxane A2, TXA2), and activated by internally applied G proteins (Galphas). Thus, the key questions that I plan to address are: What are the biophysical and pharmacological characteristics of KCa and KATP channels from CSM? Are these channels spontaneously active in resting cells? What is the mechanism of inhibition of KATP channels by ATP? What is the mechanism of action of vasoactive substances on KCa channels, are G proteins involved? Are KATP channels modulated by vasoactive substances, as well? To answer these questions, I plan: 1) to study further the biophysical and pharmacological characteristics of KCa and KATP channels; 2) to characterize the action of AgII and TXA2 on KCa channels, and to explore potential actions of these vasoactive agents on KATP channels; 3) to determine the mechanism(s) of KCa channel modulation by AgII and TXA2 (direct ligand gating, direct G protein gating "membrane delimited", and G protein mediated metabolic pathways leading to phosphorylation and/or arachidonic acid (AA) metabolism, will be explored); 4) to study if phosphorylation of dephosphorylation modify the actions of AgII and TXA2, and 5) to investigate if KCa and/or KATP channels are affected by vasorelaxants like AA and prostaglandin I2, and their mode of action. To accomplish the proposed aims, I will use a combined approach, which consists of studies using: a) the patch clamp technique (whole cell, cell attached and cell free patches) on single cells, and b) channels functionally reconstituted into bilayers. To perform patch clamp studies I have developed a single cell preparation from coronary muscle. In addition, I have succeeded in isolating a coronary plasma membrane enriched fraction which allowed to functionally reconstitute in bilayers KCa and KATP channels. These studies will help to understand how the coronary tone is regulated, how "K+ channel openers" act to alleviate cardiovascular diseases, and probably to design therapeutical treatments that reduce coronary spasm responsible for myocardial ischemia and heart dysfunction.