Nishihara, Tomonobu, Ishihara, Takeshi, Teramoto, Takayuki, Nagai, Takeharu, Matsuda, Tomoki, Kuge, Sayuri, Furuie, Hironobu
[
International Worm Meeting,
2015]
Fluorescent Ca2+ indicators have been rapidly improved for the last several decades and now are indispensable tools for cell biology, especially neuronal science, because they enable us to monitor neuronal activities. However, these fluorescent Ca2+ indicators including GCaMPs are ideal only for monitoring the activation of neurons. To understand precise functions of the neuronal network, a fluorescent Ca2+ indicator that is suitable for monitoring the neuronal inhibition would be necessary. We previously reported various "pericam" types of indicators that are based on circularly permuted green fluorescent protein (cpGFP) fused to calmodulin and calmodulin-target peptide, M13. Among these, "inverse-pericam" has unique property; the fluorescence intensity gets 7-fold dimmer upon Ca2+ binding. Although the dynamic range is relatively big among that of the reported Ca2+ indicators, it should be enlarged to effectively monitor the neuronal inhibition. To this end, we first created a large mutant library of "inverse-pericam" by iterative error-prone PCR. The mutant libraries of genetic variants of inverse-pericam indicators were expressed in the E. coli periplasm and were screened by comparing fluorescence intensity between high- and low-Ca2+ conditions. After screening of about 20,000 bacterial colonies, we found a variant, which shows nearly 20-folds dynamic range in vitro. When the inverse-pericam variant, which we named IP2.0 was expressed in HeLa cells, the fluorescence of IP2.0 was greatly quenched responding to histamine stimulation, indicating that IP2.0 is useful to detect change of the intracellular concentration of Ca2+. Then we examined whether the decrease of Ca2+ concentration during the stimulation of isoamyl alcohol, as well as the increase of Ca2+ concentration after removing isoamyl alcohol can be monitored by expressing IP2.0 in AWCON neuron of C. elegans. While the fluorescence of GCaMPs has known to be reduced slightly during the stimulation, we observed the increase of fluorescence of IP2.0 during the stimulation. These results suggest that odor stimuli decreases Ca2+ concentration in AWCON neurons and so IP2.0 will be an indispensable tool for studying neuronal inhibition.