Ca2+ Channel Inhibition Kinetics by Video Microscopy

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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.