STRUCTURE AND DYNAMICS OF CONNEXIN26 GAP JUNCTIONS

Summary

Principal Investigator: Gina E Sosinsky
Abstract: DESCRIPTION (provided by applicant): Gap junctions play dynamic roles in cellular processes, however, there is a fundamental knowledge gap in understanding how gap junction proteins, the connexins, are regulated and gated based on a structure that has rigid and flexible domains. Connexin expression and function are highly regulated and the sequence of each isoform imparts specificity ("permselectivity") to which molecules pass through the pore. The connexin hexamer (connexon or hemichannel) have three domains defined by the lipid bilayer. Two hemichannels pair at their extracellular domains to form an intercellular channel. The conserved transmembrane and extracellular domains are fairly rigid while the cytoplasmic domain is flexible. The sequence variability in the cytoplasmic domains, particularly in the C-terminus, allows for binding of partner proteins unique to each isoform. Within the context of this compartmentalized structure, our central hypothesis is that the monomer is tightly packed in its rigid domains, but flexibility in the cytoplasmic domains permit supra-molecular complexes to be formed in cells as well as binding of proteins controlling phosphorylation and gating. Connexin-opathies, hereditary human diseases, are often caused by mutations that often disrupt packing or partner interactions. For example, Cx26 mutations account for ~1/2 of cases of pre-lingual non-syndromic deafness in Caucasian populations but cases are found in populations across all continents. The proposed studies explore this hypothesis with three specific aims. (1) To investigate the stability of the transmembrane region of the Cx26 hexamer using mutations known to cause heredity deafness. These experiments will be correlated with ones probing channel function and structure. (2) To determine the 3D structure by cryo-electron microscopy (cryo-EM) and single particle reconstruction of Cx50 hemichannels. Cx50 intercellular channels serve critical functions in lens and its dysfunction leads to cataracts. It has extensive less ordered cytoplasmic domains typically not resolvable by crystallography. In this aim, single particle reconstruction is the best technique to obtain a structure of the large, full-length Cx50 hemichannel. (3) To create electron tomographic volumes of genetically labeled Cx43 intercellular channels and cytoskeletal and scaffolding proteins in situ to better understand the cytoplasmic architecture interacting with a gap junction. Cx43 contains binding domains for cytoskeletal components and the scaffolding protein, ZO-1. It is widespread through most organ systems with particularly important roles in vasculature and heart. The long-term goal is to obtain a more complete depiction of full-length connexins at the highest resolution obtainable. The approach is innovative because it uses a multi-resolution imaging strategy coordinated with biochemical and functional analyses of channels and hemichannels. The proposed research is significant because results will be useful in defining better drugs and other therapeutics that potentially ameliorate connexin-related diseases.
Funding Period: 2003-08-01 - 2016-11-30
more information: NIH RePORT

Top Publications

  1. ncbi Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel
    Derek L Beahm
    Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA
    J Biol Chem 281:7994-8009. 2006
  2. pmc Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093 0608, USA
    J Biol Chem 285:24420-31. 2010
  3. pmc Trafficking and recycling of the connexin43 gap junction protein during mitosis
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA
    Traffic 11:1471-86. 2010
  4. pmc Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels
    Atsunori Oshima
    Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo ku, Kyoto 606 8502, Japan
    J Mol Biol 405:724-35. 2011
  5. pmc Pannexin channels are not gap junction hemichannels
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA
    Channels (Austin) 5:193-7. 2011
  6. pmc Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly
    Yong Chun Yu
    Institute of Neurobiology, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China
    Nature 486:113-7. 2012
  7. pmc Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging
    Mark H Ellisman
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Methods Cell Biol 111:139-55. 2012
  8. pmc Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation
    Elena Dolmatova
    Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
    Am J Physiol Heart Circ Physiol 303:H1208-18. 2012
  9. pmc Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
    Jeffrey D Martell
    Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
    Nat Biotechnol 30:1143-8. 2012
  10. pmc Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, California, USA
    Biophys J 98:1809-19. 2010

Research Grants

  1. Mechanisms of Gap Junction Regulation
    Paul L Sorgen; Fiscal Year: 2013
  2. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
  3. Internalization of gap junctions as a regulatory mechanism of direct GJIC
    Matthias M Falk; Fiscal Year: 2013
  4. STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING
    Gina E Sosinsky; Fiscal Year: 2013
  5. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013

Detail Information

Publications22

  1. ncbi Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel
    Derek L Beahm
    Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA
    J Biol Chem 281:7994-8009. 2006
    ..This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo...
  2. pmc Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093 0608, USA
    J Biol Chem 285:24420-31. 2010
    ..We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other...
  3. pmc Trafficking and recycling of the connexin43 gap junction protein during mitosis
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA
    Traffic 11:1471-86. 2010
    ..In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit...
  4. pmc Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels
    Atsunori Oshima
    Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo ku, Kyoto 606 8502, Japan
    J Mol Biol 405:724-35. 2011
    ..Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration...
  5. pmc Pannexin channels are not gap junction hemichannels
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA
    Channels (Austin) 5:193-7. 2011
    ..Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature...
  6. pmc Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly
    Yong Chun Yu
    Institute of Neurobiology, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China
    Nature 486:113-7. 2012
    ..These results suggest a strong link between lineage-dependent transient electrical coupling and the assembly of precise excitatory neuron microcircuits in the neocortex...
  7. pmc Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging
    Mark H Ellisman
    National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Methods Cell Biol 111:139-55. 2012
    ..Here we review and discuss the current generation of genetic labels for direct protein identification by CLEM, addressing their relative strengths and weaknesses and in what experiments they would be most useful...
  8. pmc Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation
    Elena Dolmatova
    Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
    Am J Physiol Heart Circ Physiol 303:H1208-18. 2012
    ..ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury...
  9. pmc Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
    Jeffrey D Martell
    Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
    Nat Biotechnol 30:1143-8. 2012
    ..Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen...
  10. pmc Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, California, USA
    Biophys J 98:1809-19. 2010
    ..In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating...
  11. pmc The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes
    Oliver Gassmann
    Institute for Organic and Biomolecular Chemistry, University of Gottingen, Germany
    J Struct Biol 168:168-76. 2009
    ..The conductance levels of Cx26M34A were found at around 45 and 70 pS...
  12. ncbi Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels
    Jinshu Yu
    BioTechnological Center, University of Technology Dresden, Tatzberg 47 51, 01307 Dresden, Germany
    J Biol Chem 282:8895-904. 2007
    ..5 degrees rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure...
  13. pmc Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule
    Atsunori Oshima
    Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo ku, Kyoto 606 8502, Japan
    Proc Natl Acad Sci U S A 104:10034-9. 2007
    ..Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule...
  14. pmc The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA 92093 0608, USA
    Biochem J 408:375-85. 2007
    ..These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43...
  15. ncbi Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, Department of Neurosciences, University of California, San Diego, La Jolla, California 92093 0608, USA
    J Biol Chem 282:31733-43. 2007
    ..We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues...
  16. pmc The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications
    Gina E Sosinsky
    National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, 1070 Basic Science Building MC 0608, 9500 Gilman Drive, La Jolla, CA 92093 0608, USA
    J Struct Biol 161:359-71. 2008
    ....
  17. pmc Computational modeling of three-dimensional electrodiffusion in biological systems: application to the node of Ranvier
    Courtney L Lopreore
    Howard Hughes Medical Institute, La Jolla, California 92037, USA
    Biophys J 95:2624-35. 2008
    ....
  18. pmc Projection structure of a N-terminal deletion mutant of connexin 26 channel with decreased central pore density
    Atsunori Oshima
    Department of Biophysics, Faculty of Science, Kyoto University, Kyoto, Japan
    Cell Commun Adhes 15:85-93. 2008
    ..The difference map between the deletion and full-length Cx26M34A channels strongly suggests that the N-terminus of connexin contributes to the plug for the physical closure of gap junction channels...
  19. pmc Trafficking dynamics of glycosylated pannexin 1 proteins
    Daniela Boassa
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, California 92093 0608, USA
    Cell Commun Adhes 15:119-32. 2008
    ..Therefore, Panx1 has kinetics and dynamics that make it unique to serve distinct functions separate from connexin-based channels...
  20. pmc The cytoplasmic accumulations of the cataract-associated mutant, Connexin50P88S, are long-lived and form in the endoplasmic reticulum
    Alexandra Lichtenstein
    Department of Pediatrics, Section of Hematology Oncology, University of Chicago, Chicago, IL 60637 1470, USA
    Exp Eye Res 88:600-9. 2009
    ..The persistence of these particles in the lens may cause light scattering and the pulverulent cataracts observed in affected individuals...
  21. pmc Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix
    Cinzia Ambrosi
    National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California San Diego, La Jolla, California, United States of America
    PLoS ONE 8:e70916. 2013
    ..Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure. ..

Research Grants30

  1. Mechanisms of Gap Junction Regulation
    Paul L Sorgen; Fiscal Year: 2013
    ....
  2. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
    ....
  3. Internalization of gap junctions as a regulatory mechanism of direct GJIC
    Matthias M Falk; Fiscal Year: 2013
    ....
  4. STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKING
    Gina E Sosinsky; Fiscal Year: 2013
    ..We investigate the connexin43 trafficking process using an imaging based approach examining the hierarchy of connexin43 phosphorylation events and where within the cell cycle, connexin43-kinase(s) interactions occurs. ..
  5. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..