Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (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 IP(3)R(1) (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 IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used 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 fluorescence and 1.3-fold increase in calcium affinity 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 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
Calcium ; chemistry ; metabolism ; Calmodulin ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Dimerization ; Green Fluorescent Proteins ; chemistry ; genetics ; metabolism ; Histidine ; chemistry ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Myosin-Light-Chain Kinase ; chemistry ; genetics ; metabolism ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics

Corneal confocal microscopy (CCM) is an in vivo corneal imaging technique, which can directly quantify corneal nerve fibers in real time. It has the characteristics of non-invasive, objective and high sensitivity. CCM can not only be used for the diagnosis and treatment evaluation of corneal diseases, but also plays an important role in the diagnosis and prognosis evaluation of some peripheral and central nervous system diseases, such as diabetes peripheral neuropathy and Parkinson's disease. In addition, the changes of corneal nerve fibers can indirectly reflect the severity of ischemic cerebrovascular disease, and it is expected to become a noninvasive bioimaging marker of ischemic cerebrovascular disease. This article reviews CCM and its application in ischemic cerebrovascular disease, in order to provide better means for early diagnosis and prognosis evaluation of ischemic cerebrovascular disease.

Corneal confocal microscopy (CCM) is a non-invasive, simple and rapid visual corneal imaging technique, which can directly conduct real-time collection and quantitative analysis of corneal nerve fibers. Studies have shown that CCM can be used in the diagnosis and prognosis evaluation of degenerative diseases, demyelinating diseases, degenerative diseases and other types of diseases of the central nervous system. In this paper, the recent advance in CCM in neurological diseases is summarized to provide new ideas for their diagnosis and prognosis evaluation.