主催: The Japanese Pharmacological Society, The Japanese Society of Clinical Pharmacology
会議名: WCP2018 (18th World Congress of Basic and Clinical Pharmacology)
開催地: Kyoto
開催日: 2018/07/01 - 2018/07/06
Background
Cyclic adenosine monophosphate (cAMP) is a common second messenger, and known to regulate axon elongation and synaptic plasticity in nerve cell. Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in cAMP concentration are powerful tool for visualizing intracellular signaling activity. However, cAMP indicator has fewer kinds than calcium ion one. cAMP indicators with different affinities have shown different kinetics, and thus careful choice of cAMP indicator with optimal affinity and kinetics is essential to elucidate the phenomenon of interest. Therefore, we developed the newly genetically encoded green fluorescence cAMP indicator (g-CR).
Method
g-CR protein was expressed in E.coli BL21 with pCold I expression system. g-CR protein was purified with cobalt resin. g-CR properties were analyzed using fluorescence microplate reader, absorption and fluorescence spectrometer, and confocal microscope.
Primary cerebellar granule cell was made from newborn ICR mouse (P5 to P7)
Result
The fluorescence intensity of g-CR increase 2.5-fold in the presence of a saturating dose of cAMP, with excitation and emission peaks at 504nm and 513nm, respectively. We next examined the dose-response relationship of g-CR with cAMP or cGMP and calculated the dissociation constants(Kd). According to the Hill equation, the cAMP Kd=2.76µM, n=1.24 and cGMP Kd=6.6µM, n=3.13. As has been reported for many other single, circular permuted GFP (cpGFP) -based indicators, g-CR showed an increase in its fluorescence intensity as a result of pH elevation. Therefore, it is necessary to be careful interpretation or pH correction of the date obtained from imaging experiments using g-CR. The binding rate was Kon=1.83×104 M-1 s-1 and Koff=3.5×10-2 s-1. We successfully monitored the elevation of cAMP levels in primary cerebellar granule cells. Furthermore, we report that we are trying to develop pharmaceutical application using g-CR.
Conclusion
cpGFP based cAMP probe should be useful for multi-color cell imaging.
