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.
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.
|Kopanic, Jennifer L; Al-mugotir, Mona H; Kieken, Fabien et al. (2014) Characterization of the connexin45 carboxyl-terminal domain structure and interactions with molecular partners. Biophys J 106:2184-95|
|Spagnol, Gaelle; Reiling, Calliste; Kieken, Fabien et al. (2014) Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain. Biomol NMR Assign 8:263-7|
|Li, Hanjun; Spagnol, Gaelle; Naslavsky, Naava et al. (2014) TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication. J Cell Sci 127:3269-79|
|Kopanic, Jennifer L; Sorgen, Paul L (2013) Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations. Biomol NMR Assign 7:293-7|
|Kopanic, Jennifer L; Al-Mugotir, Mona; Zach, Sydney et al. (2013) An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain. Front Pharmacol 4:106|
|Grosely, Rosslyn; Kopanic, Jennifer L; Nabors, Sarah et al. (2013) Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain. J Biol Chem 288:24857-70|
|Grosely, Rosslyn; Kieken, Fabien; Sorgen, Paul L (2013) ¹H, ¹³C, and ¹?N backbone resonance assignments of the connexin43 carboxyl terminal domain attached to the 4th transmembrane domain in detergent micelles. Biomol NMR Assign 7:299-303|
|Grosely, Rosslyn; Sorgen, Paul L (2013) A history of gap junction structure: hexagonal arrays to atomic resolution. Cell Commun Adhes 20:11-20|
|Locke, Darren; Kieken, Fabien; Tao, Liang et al. (2011) Mechanism for modulation of gating of connexin26-containing channels by taurine. J Gen Physiol 138:321-39|
|Grosely, Rosslyn; Kieken, Fabien; Sorgen, Paul L (2010) Optimizing the solution conditions to solve the structure of the Connexin43 carboxyl terminus attached to the 4(th) transmembrane domain in detergent micelles. Cell Commun Adhes 17:23-33|
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