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.

Public Health Relevance

Intracellular pH is one of the most generic regulators of intercellular communication and intracellular acidification leads to closure and degradation of gap junctions, including channels formed of Cx43, the most abundant gap junction protein in the heart. The study of pH- dependent regulation of gap junctions becomes more relevant given that intracellular acidification is a major consequence of tissue ischemia. Acidification-induced uncoupling has an impact on the preservation of tissue surrounding the ischemic area in the heart and may be a key substrate for life-threatening arrhythmias during a heart attack.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Intercellular Interactions (ICI)
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Deatherage, James F
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University of Nebraska Medical Center
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Spagnol, Gaelle; Trease, Andrew J; Zheng, Li et al. (2018) Connexin43 Carboxyl-Terminal Domain Directly Interacts with ?-Catenin. Int J Mol Sci 19:
Sorgen, Paul L; Trease, Andrew J; Spagnol, Gaelle et al. (2018) Protein?Protein Interactions with Connexin 43: Regulation and Function. Int J Mol Sci 19:
Trease, Andrew J; Capuccino, Juan M V; Contreras, Jorge et al. (2017) Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization. J Mol Cell Cardiol 111:69-80
Jacobsen, Nicole L; Pontifex, Tasha K; Li, Hanjun et al. (2017) Regulation of Cx37 channel and growth-suppressive properties by phosphorylation. J Cell Sci 130:3308-3321
Li, Hanjun; Spagnol, Gaelle; Pontifex, Tasha K et al. (2017) Chemical shift assignments of the connexin37 carboxyl terminal domain. Biomol NMR Assign 11:137-141
Spagnol, Gaelle; Kieken, Fabien; Kopanic, Jennifer L et al. (2016) Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus. J Biol Chem 291:7637-50
Ambrosi, Cinzia; Ren, Cynthia; Spagnol, Gaelle et al. (2016) Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1. PLoS One 11:e0157073
Spagnol, Gaƫlle; Al-Mugotir, Mona; Kopanic, Jennifer L et al. (2016) Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms. Biopolymers 105:143-62
Bahl, Kriti; Xie, Shuwei; Spagnol, Gaelle et al. (2016) EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides. J Biol Chem 291:13465-78
Li, Hanjun; Spagnol, Gaelle; Zheng, Li et al. (2016) Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2. J Biol Chem 291:15867-80

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