CAFFEINE-ACTIVATED PLASMA MEMBRANE CATION CHANNELS

描述：（申请人摘要） 本申请的焦点在于特异性Ca 2+内流途径：一种新的， 质膜，Ca 2+渗透性，非选择性阳离子通道开放的 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，即使在任何时候只有少数几个是开放的， 它们可以对细胞内Ca 2+浓度产生很大的影响，但它们的 生理转导机制及其与 兰尼碱受体仍有待确定。第二，由于固有的 通道的特性和我们独特的成像能力，我们可以 同时记录单位电流并跟踪荧光瞬变 这是由于在通道的单次打开期间Ca 2+流入。因此可以 用于提供荧光瞬变与 未知的Ca 2+电流，如Ca 2+火花和喷烟， 从细胞内储存的Ca 2+的局部释放，并且通过扩展， 与这些事件相关的ryanodine或IP 3受体的数量。它也可以 可用于研究单个通道附近的局部Ca 2+处理。 我们将首先使用蟾蜍胃平滑肌细胞进行研究 因为我们对这些通道的生理学了解更多， 准备.我们还将使用大鼠心肌细胞。我们的实验将解决 以下问题。一个已知的单身汉和一个 通道Ca 2+电流和由此产生的荧光瞬态？这怎么能 关系可以用来确定当前潜在的Ca 2+火花？是什么 是自然细胞过程还是打开通道的内源性配体？是 质膜上的咖啡因激活的通道与 ryanodine受体？什么样的Ca 2+处理事件的环境中，一个单一的 通道显著影响细胞内Ca 2+的变化， Ca 2+荧光在一个单一的开放的通道？研究概述 这项建议应提供新的见解所涉及的机制， 兴奋-收缩偶联位于骨骼的中心， 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.