CAFFEINE-ACTIVATED PLASMA MEMBRANE CATION CHANNELS

描述：(申请人摘要) 本申请的重点是一种特定的钙离子内流途径：一种新的， 质膜，钙离子渗透性，非选择性阳离子通道，由 咖啡因的应用。咖啡因激活通道的重要性在于 在两个方面：第一，尽管只有少数几个城市同时开放， 它们可以对细胞内钙离子浓度有很大的影响，但它们的 细胞的生理转导机制及其可能的关系 兰尼定受体仍有待确定。第二，因为固有的 频道的属性和我们独特的成像能力，我们可以 同时记录单一电流并跟踪荧光瞬变 这是由于在通道的单个打开期间钙离子内流所致。因此，它可以是 用于提供荧光瞬变与 钙离子火花和喷发等基本事件的未知钙电流 细胞内钙离子的局部释放，进而延伸到细胞内 与这些事件相关的兰尼定或IP3受体的数量。它还可以 用于研究单个通道附近的局部钙离子处理。 我们将首先使用蟾蜍胃平滑肌细胞进行研究 因为我们对这些经络的生理学有更多的了解 准备工作。我们还将使用大鼠心肌细胞。我们的实验将解决 以下是一些问题。一首已知的单曲和一首 通道钙电流和由此产生的荧光瞬变？这怎么可能 关系被用来确定当前潜在的钙火花？何谓 是自然的细胞过程还是打开通道的内源性配体？是 质膜咖啡因激活通道在某种程度上与 兰尼定受体？在一个单一的环境中，有哪些钙离子处理事件 通道显著影响由此引起的细胞内钙离子和 钙离子在通道单次开放过程中的荧光？概述的研究 应为以下机制提供新的见解： 位于骨骼中心的兴奋-收缩耦合， 在正常和疾病状态下的心脏和平滑肌功能。

DESCRIPTION: (Applicant's Abstract) The focus of this application is on a specific Ca2+ influx pathway: a novel, plasma membrane, Ca2+ permeable, nonselective cation channel opened by the application of caffeine. The importance of the caffeine-activated channels lies in two areas: First, even though only of few of them are open at any one time, they can have a large effect on the intracellular Ca2+ concentration, yet their physiological transduction mechanism and their possible relationship to ryanodine receptors remain to be determined. Second, because of the inherent properties of the channel and our unique imaging capabilities, we can simultaneously record the unitary current and follow the fluorescence transient due to Ca2+ influx during a single opening of the channel. Therefore, it can be used to provide the relationship between fluorescence transients and the unknown Ca2+ current for such elementary events as Ca2+ sparks and puffs due to localized release of Ca2+ from intracellular stores and, by extension, the number of ryanodine or IP3 receptors associated with these events. It can also be used to study localized Ca2+ handling in the vicinity of a single channel. We will carry out our studies at first using toad stomach smooth muscle cells because more is known about the physiology of these channels in this preparation. We will also use rat cardiac cells. Our experiments will address the following questions. What is the exact relationship between a known single channel Ca2+ current and the resulting fluorescence transient? How can this relationship be used to determine the current underlying Ca2+ sparks? What are the natural cellular processes or endogenous ligands that open the channel? Are the plasma membrane caffeine-activated channels related in some way to ryanodine receptors? What Ca2+ handling events in the environment of a single channel significantly affect the resulting change in intracellular Ca2+ and Ca2+ fluorescence during a single opening of the channel? The studies outlined in this proposal should provide new insights into mechanisms involved in excitation-contraction coupling which is at the center of the skeletal, cardiac, and smooth muscle function in both normal and disease states.