Mechanisms of Gap Junction Regulation

Summary

Principal Investigator: Paul L Sorgen
Abstract: DESCRIPTION (provided by applicant): Connexins are integral membrane proteins that oligomerize to form intercellular gap junction channels. Ions and small molecules diffuse intercellularly through these channels, allowing individual cell events to synchronize into the functional response of an entire organ. Gap junctions mediate vitally important processes such as electrical impulse propagation, regulation of cell growth, and organ development. Moreover, mutations in a gap junction protein are linked to various inherited diseases, including nervous system disorders, deafness, cataracts, heart defects, and skin diseases. While there is considerable information regarding key interactions of connexins in the regulation of gap junction channels, the precise mechanisms that lead to channel closure and degradation have not been defined, nor have the critical accessory proteins involved been fully characterized. This information is pivotal if the role of intercellular communication in normal and diseased states is to be fully understood. The long-term goal of our work is to gain a structural and functional understanding of the mechanisms regulating gap junctions. The objective of this project is to use a multi-disciplinary approach to investigate intra- and intermolecular interactions that define the structure of the major cardiac gap junction protein connexin43 (Cx43) during pH-mediated gating and degradation. The central hypothesis for the proposed research is that Cx43 carboxyl terminal (Cx43CT) residues Y265-A305 act as a master regulatory domain that, under the appropriate conditions (e.g., intracellular acidification and/or phosphorylation), binds to a "receptor" (i.e., Cx43 cytoplasmic loop (Cx43CL)) affiliated with the pore to close the channel and then to molecular partners involved in its degradation. The study of pH-mediated Cx43 regulation is significant because intracellular acidification, which leads to closure and degradation of gap junctions, is a major consequence of tissue ischemia. In particular, acidification-induced closure and degradation of Cx43 gap junctions may be one of the causes for malignant ventricular arrhythmias during myocardial ischemia and infarction. The rationale for the proposed research is that a better understanding of the structural basis of Cx43 regulation will lead to better strategies to modulate gap junction communication that has been altered due to disease and ischemia injury. The following Specific Aims are proposed to investigate this concept: 1) To define how c-Src mediates closure of Cx43 gap junctions, 2) To determine the molecular interactions involved in Cx43 degradation, and 3) To identify molecules that can regulate junctional communication.
Funding Period: 2006-06-01 - 2015-11-30
more information: NIH RePORT

Top Publications

  1. ncbi Identification of a novel peptide that interferes with the chemical regulation of connexin43
    Junko Shibayama
    Department of Pharmacology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA
    Circ Res 98:1365-72. 2006
  2. pmc Optimizing the solution conditions to solve the structure of the Connexin43 carboxyl terminus attached to the 4(th) transmembrane domain in detergent micelles
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    Cell Commun Adhes 17:23-33. 2010
  3. pmc Mechanism for modulation of gating of connexin26-containing channels by taurine
    Darren Locke
    Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, USA
    J Gen Physiol 138:321-39. 2011
  4. pmc Characterization of the structure and intermolecular interactions between the connexin 32 carboxyl-terminal domain and the protein partners synapse-associated protein 97 and calmodulin
    Kelly Stauch
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    J Biol Chem 287:27771-88. 2012
  5. 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
  6. pmc ¹H, ¹³C, and ¹⁵N backbone resonance assignments of the connexin43 carboxyl terminal domain attached to the 4th transmembrane domain in detergent micelles
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA
    Biomol NMR Assign 7:299-303. 2013
  7. pmc Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations
    Jennifer L Kopanic
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA
    Biomol NMR Assign 7:293-7. 2013
  8. pmc A history of gap junction structure: hexagonal arrays to atomic resolution
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198 5870, USA
    Cell Commun Adhes 20:11-20. 2013
  9. pmc Gap junctions
    Morten Schak Nielsen
    Department of Biomedical Sciences and The Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
    Compr Physiol 2:1981-2035. 2012
  10. ncbi Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain
    Gaelle Spagnol
    Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA
    Biomol NMR Assign 8:263-7. 2014

Detail Information

Publications29

  1. ncbi Identification of a novel peptide that interferes with the chemical regulation of connexin43
    Junko Shibayama
    Department of Pharmacology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA
    Circ Res 98:1365-72. 2006
    ..These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias...
  2. pmc Optimizing the solution conditions to solve the structure of the Connexin43 carboxyl terminus attached to the 4(th) transmembrane domain in detergent micelles
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    Cell Commun Adhes 17:23-33. 2010
    ..Additionally, the optimal solution conditions for solving the NMR solution structure include 10% 2,2,2 trifluoroethanol and removal of the 2nd extracellular loop (G178-V196)...
  3. pmc Mechanism for modulation of gating of connexin26-containing channels by taurine
    Darren Locke
    Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, USA
    J Gen Physiol 138:321-39. 2011
    ..These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function...
  4. pmc Characterization of the structure and intermolecular interactions between the connexin 32 carboxyl-terminal domain and the protein partners synapse-associated protein 97 and calmodulin
    Kelly Stauch
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    J Biol Chem 287:27771-88. 2012
    ..Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis...
  5. 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...
  6. pmc ¹H, ¹³C, and ¹⁵N backbone resonance assignments of the connexin43 carboxyl terminal domain attached to the 4th transmembrane domain in detergent micelles
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA
    Biomol NMR Assign 7:299-303. 2013
    ....
  7. pmc Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations
    Jennifer L Kopanic
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA
    Biomol NMR Assign 7:293-7. 2013
    ....
  8. pmc A history of gap junction structure: hexagonal arrays to atomic resolution
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198 5870, USA
    Cell Commun Adhes 20:11-20. 2013
    ..Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology...
  9. pmc Gap junctions
    Morten Schak Nielsen
    Department of Biomedical Sciences and The Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
    Compr Physiol 2:1981-2035. 2012
    ..This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease...
  10. ncbi Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain
    Gaelle Spagnol
    Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA
    Biomol NMR Assign 8:263-7. 2014
    ..A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation. ..
  11. pmc Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain
    Rosslyn Grosely
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    J Biol Chem 288:24857-70. 2013
    ....
  12. pmc Carboxy terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37-mediated suppression of insulinoma cell proliferation
    Tasha K Nelson
    Department of Physiology, University of Arizona, Tucson, Arizona and
    Am J Physiol Cell Physiol 305:C1246-56. 2013
    ....
  13. pmc Mechanism for the selective interaction of C-terminal Eps15 homology domain proteins with specific Asn-Pro-Phe-containing partners
    Fabien Kieken
    Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198 5870, USA
    J Biol Chem 285:8687-94. 2010
    ..Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs...
  14. pmc Characterization of the structure and intermolecular interactions between the connexin40 and connexin43 carboxyl-terminal and cytoplasmic loop domains
    Denis Bouvier
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    J Biol Chem 284:34257-71. 2009
    ....
  15. pmc Effect of charge substitutions at residue his-142 on voltage gating of connexin43 channels
    Junko Shibayama
    Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA
    Biophys J 91:4054-63. 2006
    ..Some of the structural constrains of fast voltage gating may be shared with those involved in the chemical gating of Cx43...
  16. ncbi Characterization of the pH-dependent interaction between the gap junction protein connexin43 carboxyl terminus and cytoplasmic loop domains
    Bethany J Hirst-Jensen
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    J Biol Chem 282:5801-13. 2007
    ....
  17. ncbi EHD1 and Eps15 interact with phosphatidylinositols via their Eps15 homology domains
    Naava Naslavsky
    Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198 5870, USA
    J Biol Chem 282:16612-22. 2007
    ....
  18. ncbi EH domain of EHD1
    Fabien Kieken
    Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA
    J Biomol NMR 39:323-9. 2007
    ..While the overall structure resembles that of the second of the three N-terminal Eps15 EH domains, potentially significant differences in surface charge and the structure of the tripeptide-binding pocket are discussed...
  19. pmc Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC
    Joell L Solan
    Molecular Diagnostics Program and 2Proteomics Shared Resource, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
    J Cell Biol 179:1301-9. 2007
    ..Thus, phosphorylation at S365 can serve a "gatekeeper" function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance...
  20. pmc Purification and reconstitution of the connexin43 carboxyl terminus attached to the 4th transmembrane domain in detergent micelles
    Admir Kellezi
    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA
    Protein Expr Purif 59:215-22. 2008
    ....
  21. pmc Structural changes in the carboxyl terminus of the gap junction protein connexin 40 caused by the interaction with c-Src and zonula occludens-1
    Denis Bouvier
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    Cell Commun Adhes 15:107-18. 2008
    ..This result differs from when the same experiment was performed with Cx43CT, suggesting different mechanisms of regulation exist between connexin isoforms, even when involving the same molecular partners...
  22. pmc Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction
    Fabien Kieken
    Department of Cardiology, Center for Life Sciences 9 913, Beth Israel Deaconess Medical Center, 3 Blackfan Circle, Boston, MA 02115, USA
    Circ Res 104:1103-12. 2009
    ..Therefore shifts in Cx43 protein partners may underlie, in part, arrhythmogenesis in the post-MI heart...
  23. pmc Eps15 homology domain 1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol-(4,5)-bisphosphate and are required for efficient recycling
    Marko Jovic
    Department of Biochemistry and Molecular Biology, and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198 5870, USA
    Mol Biol Cell 20:2731-43. 2009
    ....
  24. pmc Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides
    Vandana Verma
    Center for Arrhythmia Research, University of Michigan Medical School, 5025 Venture Dr, Ann Arbor MI 48104, USA
    Circ Res 105:176-84. 2009
    ..Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function...
  25. ncbi (1)H, (13)C, and (15)N backbone resonance assignments of the carboxyl terminal domain of Connexin40
    Denis Bouvier
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, 68198, USA
    Biomol NMR Assign 1:155-7. 2007
    ..The assignments of an important Connexin40 regulatory domain, the carboxyl terminus, will aid in identifying the types of inter- and intramolecular interactions that affect channel activity...
  26. pmc Structural insight into the interaction of proteins containing NPF, DPF, and GPF motifs with the C-terminal EH-domain of EHD1
    Fabien Kieken
    Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198 5870, USA
    Protein Sci 18:2471-9. 2009
    ..The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues...
  27. pmc Characterization of the connexin45 carboxyl-terminal domain structure and interactions with molecular partners
    Jennifer L Kopanic
    Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska
    Biophys J 106:2184-95. 2014
    ..This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites...

Research Grants30

  1. Connexin Distribution in Physiological Versus Pathological Cardiac Hypertrophy
    Michael R Zile; Fiscal Year: 2013
    ..pathological hypertrophy, with ex- tensively characterized cytoskeletal properties in each setting. ..