Ca2+ Channel Inhibition Kinetics by Video Microscopy

项目标题：视频显微镜下的钙通道抑制动力学P1：Stephen J.Morris，UM-KC分子生物学和生物化学。建议：9907571位于细胞膜上的钙通道控制大量的细胞活动。在神经和内分泌细胞中，这些通道的开放标志着神经递质或激素的释放。CA2通道受多种质膜受体的抑制。在许多神经和内分泌细胞中发现的多巴胺D2受体可以通过阻断钙通道来非常迅速地抑制神经调节剂的释放。这项提议有两个目标：首先，开发一种基于成像的方法来测量钙通道动力学的某些快速方面，然后使用该方法来研究多巴胺D2受体对电压激活的钙通道的调节。一个特殊设计的显微镜将被用来捕捉放置在活细胞中的探针的荧光，这将向我们展示当活细胞发生时，活细胞中钙水平的动态变化。这种实时的、基于成像的方法将间接测量细胞内钙水平变化时的钙通道活动。到目前为止，这类实验都是通过膜片钳技术进行的。我们的方法不会替代膜片钳方法，而是作为对全细胞膜片钳的补充，并在某些研究中作为一种可行的替代方法。与斑片cAMP实验者使用的药理学工具相同，我们将使用相同的药理学工具来确定质膜通道是所见变化的来源，而不是从细胞内储存释放。将确定受体抑制了几种亚型通道中的哪些。最后将测量D2受体抑制钙通道所需的时间，以毫秒为单位。该方法为超过50毫秒一半的事件提供了几个优点，尽管它与全细胞膜片钳一样昂贵，目前无法测量10-20毫秒的事件。它对钙动力学的应用只是众多应用中的一个。只有可用的荧光探针限制了这种可能性。每天都有新的探头上市。

Project title: Ca 2+ Channel Inhibition Kinetics by Video MicroscopyP1: Stephen J. Morris, Molecular Biology and Biochemistry, UM-KC.Proposal: 9907571Ca2 + channels located in the cell plasma membrane control a large number of cell activities. In nerve and endocrine cells, opening of these channels signals the release of neurotransmitters or hormones. CA2+ channels are inhibited by a number of plasma membrane receptors. The dopamine D2 receptor found in a number of nerves and endocrine cells can very rapidly inhibit neuromodulator release by blocking Ca2+ channels. This proposal has two goals: first to develop an imaging-based method for measuring certain rapid aspects of Ca2+ channel kinetics, then to use the method to study dopamine D2 receptor regulation of voltage activated Ca2+ channels.A specially designed microscope will be used to capture the fluorescent light from probes placed in living cells which will show us the dynamic changes in the Ca2+ levels of living cells, as they happen. This real-time, imaging-based method would measure Ca2+ channel activity indirectly as changes in intracellular Ca2+ levels. Up to now, this type of experiment has been done by patch clamp methodology. Our approach would not be a replacement for patch clamp methods, but rather be used as a complement to, and for some studies, a viable alternative to whole cell patch clamp.The same pharmacological tools as used by patch camp experimenters will be used to establish that plasma membrane channels were the sources for changes seen, and not release from intracellular stores. Which of several sub-types of channels are inhibited by the receptor will be identified. Finally the time, in milliseconds, required for the D2 receptors to inhibit Ca2+ channels will be measured.The method offers several advantages for events which have half-times longer than 50 msec, although it is as expensive as that for whole cell patch clamp, and presently cannot measure 10 - 20 msec events. Its use for Ca2+ kinetics is only one of many. Only the fluorescent probes available limit the possibilities. New probes are marketed daily.