Abstract
A prevailing view regarding the regulation of connexin43 (Cx43) gap junction channels is that, upon intracellular acidification, the carboxyl-terminal domain (Cx43CT) moves toward the channel opening to interact with specific residues acting as a receptor site. Previous studies have demonstrated a direct, pH-dependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on alpha-helical formation for the peptide in response to acidification; more recent studies have shown that acidification also induces Cx43CT dimerization. Whether Cx43CT dimerization is an important structural component in Cx43 regulation remains to be determined. Here we used an assortment of complimentary biophysical techniques to characterize the binding of Cx43CT or its mutants to itself and/or to a more native-like Cx43CL construct (Cx43CL(100-155), residues 100-155). Our studies expand the observation that specific Cx43CT domains are important for dimerization. We further show that properties of the Cx43CL(100-155) are different from those of the Cx43CL peptide; solvent acidification leads to Cx43CL(100-155) oligomerization and a change in the stoichiometry and binding affinity for the Cx43CT. Homo-Cx43CT and Cx43CL(100-155) oligomerization as well as the Cx43CT/Cx43CL(100-155) interaction can occur under in vivo conditions; moreover, we show that Cx43CL(100-155) strongly affects resonance peaks corresponding to Cx43CT residues Arg-376-Asp-379 and Asn-343-Lys-346. Overall, our data indicate that many of the sites involved in Cx43CT dimerization are also involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels.
Highlights
Gap junctions are integral membrane proteins that enable direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells
We further show that properties of the Cx43CL100–155 are different from those of the Cx43 cytoplasmic loop (Cx43CL) peptide; solvent acidification leads to Cx43CL100–155 oligomerization and a change in the stoichiometry and binding affinity for the Cx43 carboxyl-terminal domain (Cx43CT)
Our data indicate that many of the sites involved in Cx43CT dimerization are involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels
Summary
Cx43, connexin; Cx43CT, connexin carboxyl terminus; Cx43CL, connexin cytoplasmic loop; BS3, bis(sulfosuccinimidyl suberate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; HSQC, heteronuclear single-quantum correlation; PBS, phosphate-buffered saline; SPR, surface plasmon resonance; ZO-1, zonula ocludens-1; MTSL, 1-oxyl-2,2,5,5-tetramethyl-D-pyrroline-3methylmethanethiosulfonate. Co-expression of the Cx43CT as a separate fragment partially recovered this function, demonstrating the possible involvement of intramolecular interactions in pH gating [20]. These data led to the notion that pH gating results from a “particle-receptor” interaction, modeled after the “ball-and-chain” mechanism of voltage-dependent inactivation [21]. In this study we have used chemical cross-linking, sedimentation equilibrium, surface plasmon resonance, yeast two-hybrid, and NMR to characterize molecular events associated with chemical regulation of Cx43 Using both Cx43CT and Cx43CL wild-type constructs and a collection of Cx43CT mutants, we demonstrate that specific domains of the Cx43CT are important for pH-dependent dimerization. We hypothesize that the pH-dependent structural modification of both the Cx43CT and Cx43CL domains is necessary for their association, which eventually leads to the closure of Cx43 gap junctions in response to low pH
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
