Ca2+ Channel Inhibition Kinetics by Video Microscopy

通过视频显微镜观察 Ca2 通道抑制动力学

• 批准号: 9907571
• 负责人: Stephen Morris
• 金额: $ 2万
• 依托单位: University of Missouri-Kansas City
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2001-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9907571&HistoricalAwards=false
• 关键词: Ca2+; Channel; Inhibition; Kinetics; Video

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.

项目名称：通过视频显微镜进行 Ca 2+ 通道抑制动力学P1：Stephen J. Morris，分子生物学和生物化学，UM-KC。提案：9907571Ca2+ 通道位于细胞质膜中控制大量细胞活动。在神经和内分泌细胞中，这些通道的打开标志着神经递质或激素的释放。 CA2+ 通道受到许多质膜受体的抑制。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 Ca2+ 通道来非常快速地抑制神经调节剂的释放。该提案有两个目标：首先开发一种基于成像的方法来测量 Ca2+ 通道动力学的某些快速方面，然后使用该方法研究多巴胺 D2 受体对电压激活的 Ca2+ 通道的调节。将使用专门设计的显微镜来捕获放置在活细胞中的探针发出的荧光将向我们展示活细胞 Ca2+ 水平发生的动态变化。这种基于成像的实时方法将间接测量 Ca2+ 通道活性作为细胞内 Ca2+ 水平的变化。到目前为止，此类实验都是通过膜片钳方法完成的。我们的方法不会取代膜片钳方法，而是用作全细胞膜片钳方法的补充，并且对于某些研究来说，是全细胞膜片钳的可行替代方案。将使用与膜片营实验人员使用的相同药理学工具来建立质膜通道是所见变化的来源，而不是从细胞内储存中释放。 将鉴定几种通道亚型中的哪一种被受体抑制。最后，将测量 D2 受体抑制 Ca2+ 通道所需的时间（以毫秒为单位）。该方法为半时长超过 50 毫秒的事件提供了几个优点，尽管它与全细胞膜片钳的方法一样昂贵，目前无法测量 10 - 20 毫秒事件。它在 Ca2+ 动力学中的应用只是众多应用之一。只有可用的荧光探针限制了可能性。每天都有新的探头上市。