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：视频显微镜下ca2 +通道抑制动力学[1]:Stephen J. Morris，分子生物学与生物化学，UM-KC。建议：位于细胞膜上的9907571Ca2 +通道控制着大量的细胞活动。在神经和内分泌细胞中，这些通道的打开表明神经递质或激素的释放。CA2+通道被许多质膜受体抑制。多巴胺D2受体存在于许多神经和内分泌细胞中，可以通过阻断Ca2+通道非常迅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，然后使用该方法来研究多巴胺D2受体对电压激活Ca2+通道的调节。一种特殊设计的显微镜将被用来捕捉从放置在活细胞中的探针发出的荧光，这将向我们展示活细胞中Ca2+水平的动态变化。这种实时的、基于成像的方法将通过细胞内Ca2+水平的变化间接测量Ca2+通道活性。到目前为止，这类实验都是用膜片钳法进行的。我们的方法不会取代膜片钳方法，而是作为补充，在一些研究中，作为全细胞膜片钳的可行替代方法。与补丁营实验人员使用的药理学工具相同，将用于确定质膜通道是所见变化的来源，而不是细胞内储存的释放。将确定哪些亚类型的通道被受体抑制。最后，D2受体抑制Ca2+通道所需的时间（以毫秒为单位）将被测量。虽然该方法与全细胞膜片钳一样昂贵，而且目前还不能测量10 - 20毫秒的事件，但该方法对于时间比50毫秒长一半的事件具有几个优点。它在Ca2+动力学中的应用只是众多应用中的一种。只有荧光探针限制了这种可能性。每天都有新的探针上市。