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7 Janelia Publications

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    06/23/20 | Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+ channels in nanodisc
    Gao S, Valinsky WC, On NC, Houlihan PR, Qu Q, Liu L, Pan X, Clapham DE, Yan N
    Proceedings of the National Academy of Sciences of the U.S.A.. 2020 Jun 23;117(25):14187-93. doi: 10.1073/pnas.1922903117

    NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure–function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV–bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.

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    09/04/19 | Isomeric tuning yields bright and targetable red Ca indicators.
    Deo C, Sheu S, Seo J, Clapham DE, Lavis LD
    Journal of the American Chemical Society. 2019 Sep 04;141(35):13734-13738. doi: 10.1021/jacs.9b06092

    Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca fluxes in the primary cilium.

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    01/18/19 | Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.
    Gao R, Asano SM, Upadhyayula S, Pisarev I, Milkie DE, Liu T, Singh V, Graves AR, Huynh GH, Zhao Y, Bogovic JA, Colonell J, Ott CM, Zugates CT, Tappan S, Rodriguez A, Mosaliganti KR, Sheu S, Pasolli HA, et al
    Science (New York, N.Y.). 2019 Jan 18;363(6424):eaau8302. doi: 10.1126/science.aau8302

    Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

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    11/11/18 | Cryo-EM structure of the receptor-activated TRPC5 ion channel at 2.9 angstrom resolution.
    Jingjing Duan , Jian Li , Gui-Lan Chen , Bo Zeng , Kechen Xie , Xiaogang Peng , Wei Zhou , Jianing Zhong , Yixing Zhang , Jie Xu , Changhu Xue , Lan Zhu , Wei Liu , Xiao-Li Tian , Jianbin Wang , David E. Clapham , Zongli Li , Jin Zhang

    The transient receptor potential canonical subfamily member 5 (TRPC5) is a non-selective calcium-permeant cation channel. As a depolarizing channel, its function is studied in the central nervous system and kidney. TRPC5 forms heteromultimers with TRPC1, but also forms homomultimers. It can be activated by reducing agents through reduction of the extracellular disulfide bond. Here we present the 2.9 Å resolution electron cryo-microscopy (cryo-EM) structure of TRPC5. The structure of TRPC5 in its apo state is partially open, which may be related to the weak activation of TRPC5 in response to extracellular pH. We also report the conserved negatively charged residues of the cation binding site located in the hydrophilic pocket between S2 and S3. Comparison of the TRPC5 structure to previously determined structures of other TRPC and TRP channels reveals differences in the extracellular pore domain and in the length of the S3 helix. Together, these results shed light on the structural features that contribute to the specific activation mechanism of the receptor-activated TRPC5.

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    08/14/18 | Structure of the mammalian TRPM7, a magnesium channel required during embryonic development.
    Duan J, Li Z, Li J, Hulse RE, Santa-Cruz A, Valinsky WC, Abiria SA, Krapivinsky G, Zhang J, Clapham DE
    Proceedings of the National Academy of Sciences of the United States of America. 2018 Aug 14;115(35):E8201-10. doi: 10.1073/pnas.1810719115

    The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg and Zn Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg ions occupying the center of the conduction pore. In high [Mg], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.

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    08/06/18 | Structure of the mouse TRPC4 ion channel.
    Duan J, Li J, Zeng B, Chen G, Peng X, Zhang Y, Wang J, Clapham DE, Li Z, Zhang J
    Nature Communications. 2018 Aug 06;9(1):3102. doi: 10.1038/s41467-018-05247-9

    Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels.

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    07/13/18 | Cryo-EM structure of the polycystin 2-l1 ion channel.
    Hulse RE, Li Z, Huang RK, Zhang J, Clapham DE
    eLife. 2018 Jul 13;7:. doi: 10.7554/eLife.36931

    We report the near atomic resolution (3.3 Å) of the human polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1.

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