title: pH-dependent dimerization of the carboxyl terminal domain of Cx43.
abstract: Previous studies have demonstrated that the carboxyl terminus of the gap junction protein Cx43 (Cx43CT) can act as an independent, regulatory domain that modulates intercellular communication in response to appropriate chemical stimuli. Here, we have used NMR, chemical cross-linking, and analytical ultracentrifugation to further characterize the biochemical and biophysical properties of the Connexin43 carboxyl terminal domain (S255-I382). NMR-diffusion experiments at pH 5.8 suggested that the Connexin43 carboxyl terminus (CX43CT) may have a molecular weight greater than that of a monomer. Sedimentation equilibrium and cross-linking data demonstrated a predominantly dimeric state for the Cx43CT at pH 5.8 and 6.5, with limited dimer formation at a more neutral pH. NMR-filtered nuclear Overhauser effect studies confirmed these observations and identified specific areas of parallel orientation within Cx43CT, likely corresponding to dimerization domains. These regions included a portion of the SH3 binding domain, as well as two fragments previously found to organize in alpha-helical structures. Together, these data show that acidification causes Cx43CT dimer formation in vitro. Whether dimer formation is an important structural component of the regulation of Connexin43 channels remains to be determined. Dimerization may alter the affinity of Cx43CT regions for specific molecular partners, thus modifying the regulation of gap junction channels.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, 68198, USA. psorgen@unmc.edu
authors.forename: Paul L
authors.initials: PL
authors.lastname: Sorgen
authors.forename: Heather S
authors.initials: HS
authors.lastname: Duffy
authors.forename: David C
authors.initials: DC
authors.lastname: Spray
authors.forename: Mario
authors.initials: M
authors.lastname: Delmar
mesh_terms: D000818:Animals; D018031:Connexin 43 / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D014461:Ultracentrifugation; D018909:src Homology Domains
chemical_list: D018031:Connexin 43; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins