Quantitative analysis of Ca 2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca 2+ -dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca 2+ binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca 2+ release kinetics with an off-rate estimated at around 700 s −1 and appropriate Ca 2+ binding affinity, likely attributable to local Ca 2+ -induced conformational changes around the designed Ca 2+ binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca 2+ dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca 2+ in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca 2+ release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca 2+ homeostasis.

Summary The precise spatiotemporal characteristics of subcellular calcium (Ca 2+ ) transients are critical for the physiological processes. Here we report a green Ca 2+ sensor called “G-CatchER + ” using a protein design to report rapid local ER Ca 2+ dynamics with significantly improved folding properties. G-CatchER + exhibits a superior Ca 2+ on rate to G-CEPIA1er and has a Ca 2+ -induced fluorescence lifetimes increase. G-CatchER + also reports agonist/antagonist triggered Ca 2+ dynamics in several cell types including primary neurons that are orchestrated by IP 3 Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER + -JP45), we report a rapid local Ca 2+ release that is likely due to calsequestrin. Transgenic expression of G-CatchER + in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca 2+ dynamics. G-CatchER + will be an invaluable tool to examine local ER/SR Ca 2+ dynamics and facilitate drug development associated with ER dysfunction.

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Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164–186) of Cx45 using a peptide model. The strong binding (Kd ~ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM’s modulation of connexins.

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (Ig V ) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the ␤-sandwich-like Ig V domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED ␤-strands and the other involving GFCCC؆ ␤-strands, with the former burying one prominent glycosylation site. These structures raise ques- The human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) 2 is involved in cell adhesion, proliferation, and immune response (1, 2). It also is implicated in cancer angiogenesis, progression, and metastasis (3). More specifically, it is known that CEACAM1 is a negative-regulator of cell proliferation and is down-regulated in some tumor cells (4 -10). Yet, CEACAM1 expression is reported to protect tumor cells from killing by immune cells (11)(12)(13)(14) and it has been found that the high expression level is associated with a number of other cancers (15)(16)(17)(18)(19). It is likely that this complex set of effects arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other members of the CEACAM superfamily. The N-terminal Ig variable (Ig V ) domain of CEACAM1 has been suggested to be the basis of cis and trans homo-dimer formation (20), as well as interactions with other molecules (21-24). There are crystal structures of the Ig V domain expressed in Escherichia coli, which have led to the suggestion of two distinct dimerization interfaces (24, 25). However, examination of the interfaces suggests that the extensive glycosylation found in native material would inhibit dimer formation in one of these cases. This raises questions about the actual type of dimer found in solution, with and without glycosylation. Here, we use NMR methods to examine dimer structures in solution using a non-glycosylated form expressed in E. coli, and several glycosylated variants expressed in HEK293 cells.Dimerization of cell surface molecules is a well accepted mechanism for transmitting signals from the cell surface to the interior (26). In the case of CEACAM1 it is believed that modulation of intercellular adhesion as well as transmission of signals to the cell interior involves switching between cis and trans dimerization interactions. The full-length CEACAM1 molecule is large, sharing a topology with many other cell-surface signaling molecules; the extracellular domain is composed of one Ig V -like domain and typically 3 Ig C2 -like dom...

Background:The calcium-sensing receptor (CaSR) senses alterations in extracellular Ca 2ϩ mainly through its extracellular domain (ECD). Results: Interactions of ligands with the CaSR were characterized using purified ECD and biophysical techniques. Conclusion: Ca 2ϩ and L-Phe bind to both the complex and high mannose forms of the CaSR ECDs and increase binding affinities for each other. Significance: Our findings will provide an essential tool for future structural studies in GPCRs.

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change.

Multi-scale calcium (Ca 2+ )d ynamics,e xhibiting wide-ranging temporal kinetics,constitutes aubiquitous mode of signal transduction. We report an ovel endoplasmicreticulum (ER)-targeted Ca 2+ indicator,R -CatchER,w hich showed superior kinetics in vitro (k off ! 2 10 3 s À1 ,k on ! 7 10 6 M À1 s À1 )a nd in multiple cell types.R -CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints,e nabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose ap rinciple to rationally design genetically encoded Ca 2+ indicators with as ingle Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore.T he design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range.Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery.