Genomes and Genes
STRUCTURE AND DYNAMICS OF CONNEXIN26 GAP JUNCTIONS
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
- Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channelDerek 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...
- Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each otherCinzia 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...
- Trafficking and recycling of the connexin43 gap junction protein during mitosisDaniela 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...
- Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channelsAtsunori 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...
- Pannexin channels are not gap junction hemichannelsGina 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...
- Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assemblyYong 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...
- Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imagingMark 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...
- Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activationElena 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...
- Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopyJeffrey 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...
- Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stabilityCinzia 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...
- The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranesOliver 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...
- Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannelsJinshu 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...
- Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibuleAtsunori 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...
- The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodiesGina 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...
- Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membraneDaniela 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...
- The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applicationsGina 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....
- Computational modeling of three-dimensional electrodiffusion in biological systems: application to the node of RanvierCourtney L Lopreore
Howard Hughes Medical Institute, La Jolla, California 92037, USA
Biophys J 95:2624-35. 2008....
- Projection structure of a N-terminal deletion mutant of connexin 26 channel with decreased central pore densityAtsunori 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...
- Trafficking dynamics of glycosylated pannexin 1 proteinsDaniela 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...
- The cytoplasmic accumulations of the cataract-associated mutant, Connexin50P88S, are long-lived and form in the endoplasmic reticulumAlexandra 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...
- Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helixCinzia 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. ..
- Mechanisms of Gap Junction RegulationPaul L Sorgen; Fiscal Year: 2013....
- TSH RECEPTOR MULTIMERIZATIONTERRY FRANCIS DAVIES; Fiscal Year: 2013....
- Internalization of gap junctions as a regulatory mechanism of direct GJICMatthias M Falk; Fiscal Year: 2013....
- STRUCTURAL ANALYSIS OF GAP JUNCTION TRAFFICKINGGina 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. ..
- 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. ..