Imaging Airway Myocyte Ca2+ Signaling in Living Animals

University Of Chicago, Chicago IL

Investigators

Abstract

[unreadable] DESCRIPTION (provided by applicant): Intracellular free calcium ion concentration ([Ca2+]i) controls a number of intracellular signaling systems, and as such is of interest to researchers who study those systems or their downstream effector systems. Other studies have demonstrated that protein-protein approximation can be detected by reconstitution of luciferase function when N- and C-terminal portions of either firefly or Renilla luciferase are each fused to one of another pair of interacting proteins. Based on this background information, we conceived, designed, implemented, and tested a novel split synthetic Renilla luciferase [Ca2+] reporter system, currently realized as a mammalian expression plasmid with CMV promoter that drives expression of a chimeric cDNA encoding a single fusion protein. We found that the luciferase activity of our probe falls substantially with increasing free [Ca2+] in a physiologically relevant range. Additional experiments are planned to more fully evaluate the probe's sensitivity to other ions; to ATP concentration; and to ambient temperature in cell lysates. In addition, we plan to evaluate whether the probe can detect intracellular calcium concentration in living cells in culture, tissue origins, and eventually in cells in living animals. [unreadable] [unreadable] [unreadable]