Genetically encoded Ca indicators(GECI)are important tools that enable the measurement of Ca dynamics for tiss...
Yingxiao Chen#,Lin Miao#,Xianqiang Song#,Sheng Ye,Yinglong Tang,Yan Chang,Rong-Guang Zhang and Guangju Ji National Laboratory of Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences, Beijing,China
Genetically encoded Ca^(2+) indicators(GECI)are important for the measurement of Ca^(2+) in vivo.GCaMP2,a widelyused GECI,has recently been iteratively improved.Among the improved variants,GCaMP3 exhibits significantly better fluorescent intensity.In this study,we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ.GCaMPJ has a 1.5-fold increase in fl uorescence and 1.3-fold increase in calcium affi nity over GCaMP3.Upon Ca^(2+) binding,GCaMP3 exhibits both monomeric and dimeric forms.The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance.However,GCaMPJ seldom forms dimers under conditions similar to GCaMP3.St ructural and mutagenesis studies on Tyr-380 confi rmed its importance in blocking the cpEGFPβ-barrel holes.Our study proposes an efficient tool for mapping Ca^(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
Yingxiao ChenXianqiang SongSheng YeLin MiaoYun ZhuRong-Guang ZhangGuangju Ji
Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate(IP3)-induced Ca2+release(IICR)via IP3R1,but the mechanism remains largely unexplored.Using extracellular ATP to induce intracellular calcium transient as an IICR model,Ca2+image,pull down assay,and Western blotting experiments were carried out in the present study.We found that extracellular ATP induced calcium transient via IP3Rs(IICR)and the IICR were markedly decreased in ERp44 overexpressed Hela cells.The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331–377)mutants of ERp44 on IICR were significantly decreased compared with ERp44.However,the binding capacity of ERp44 to L3V domain of IP3R1(1L3V)was enhanced by ERp44 C160S/C212S mutation.Taken together,these results suggest that the mutants of ERp44,C160/C212,can more tightly bind to IP3R1 but exhibit a weak inhibition of IP3R1 channel activity in Hela cells.
Congyan PanJi ZhengYanyun WuYingxiao ChenLikun WangZhansong ZhouWenxuan YinGuangju Ji
