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      A cryo-EM-based model of phosphorylation- and FKBP12.6-mediated allosterism of the cardiac ryanodine receptor.

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          Abstract

          Type 2 ryanodine receptors (RyR2s) are calcium channels that play a vital role in triggering cardiac muscle contraction by releasing calcium from the sarcoplasmic reticulum into the cytoplasm. Several cardiomyopathies are associated with the abnormal functioning of RyR2. We determined the three-dimensional structure of rabbit RyR2 in complex with the regulatory protein FKBP12.6 in the closed state at 11.8 Å resolution using cryo-electron microscopy and built an atomic model of RyR2. The heterogeneity in the data set revealed two RyR2 conformations that we proposed to be related to the extent of phosphorylation of the P2 domain. Because the more flexible conformation may correspond to RyR2 with a phosphorylated P2 domain, we suggest that phosphorylation may set RyR2 in a conformation that needs less energy to transition to the open state. Comparison of RyR2 from cardiac muscle and RyR1 from skeletal muscle showed substantial structural differences between the two, especially in the helical domain 2 (HD2) structure forming the Clamp domain, which participates in quaternary interactions with the dihydropyridine receptor and neighboring RyRs in RyR1 but not in RyR2. Rigidity of the HD2 domain of RyR2 was enhanced by binding of FKBP12.6, a ligand that stabilizes RyR2 in the closed state. These results help to decipher the molecular basis of the different mechanisms of activation and oligomerization of the RyR isoforms and could be extended to RyR complexes in other tissues.

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          Most cited references46

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          FREALIGN: high-resolution refinement of single particle structures.

          The refinement of three-dimensional reconstructions and correction for the contrast transfer function of the microscope are important steps in the determination of macromolecular structures by single particle electron microscopy. The algorithms implemented in the computer program FREALIGN are optimized to perform these tasks efficiently. A general overview and details on how to use FREALIGN are provided. The program is free and available for download on the author's web page.
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            Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution

            The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.
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              Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators.

              The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.
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                Author and article information

                Journal
                Sci Signal
                Science signaling
                American Association for the Advancement of Science (AAAS)
                1937-9145
                1945-0877
                May 23 2017
                : 10
                : 480
                Affiliations
                [1 ] Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298, USA.
                [2 ] University of Virginia, Charlottesville, VA 22903, USA.
                [3 ] Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298, USA. montserrat.samso@vcuhealth.org.
                Article
                10/480/eaai8842
                10.1126/scisignal.aai8842
                28536302
                aaf0a749-f1c9-49e2-8330-73be804787ca
                History

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