This proposal describes the development and use of a novel sensor for cAMP to examine the interplay between the Ca2+ and cAMP signaling pathways. Dynamic Ca2+ signaling pathways are intimately related to cAMP signaling pathways, with numerous avenues for co-regulation. However, the current method for measuring cAMP in vivo only allows identification of slow changes in cAMP throughout the cytoplasm. A novel cAMP sensor, a modified CNG channel, has largely been developed. Optical and electrophysiological techniques will be used to address the following questions in the further development and use of this novel sensor. 1. Can Ca2+ flux through the olfactory CNG channel pore generate local [Ca2+] high enough to inhibit channel activity or is Ca2+ regulation of the channel dependent upon other Ca2+ sources? Can the disruption of the Ca 2+- calmodulin binding site remove Ca2+ effects on channel activity? 2. Can the olfactory CNG channel be used as a real- time measure of adenylyl cyclase (AC) activity in the human embryonic kidney cell line. HEK293, and the C6-2B glioma cell line (which expresses predominantly a Ca2+ inhibitable cyclase)? If so, the sensor will be used to investigate the interplay between AC and phosphodiesterase (PDE) activities in regulating cAMP levels. 3. Are Ca2+ and cAMP signaling interrelated in pituitary GH3 cell line? What are the critical molecular components contributing to their interactions in these excitable cells? Does [cAMP] oscillate in conjunction with [Ca2+]?
