Optimizing the Solution Conditions to Solve the Structure of the Connexin43 Carboxyl Terminus Attached to the 4^th Transmembrane Domain in Detergent Micelles

Abstract

pH-mediated gating of Cx43 channels following an ischemic event is believed to contribute to the development of lethal cardiac arrhythmias. Studies using a soluble version of the Cx43 carboxyl-terminal domain (Cx43CT; S255–I382) have established the central role it plays in channel regulation; however, research in the authors' laboratory suggests that this construct may not be the ideal model system. Therefore, we have developed a more ‘native-like’ construct (Cx43CT attached to the 4th transmembrane domain [TM4-Cx43CT; G178–I382]) than the soluble Cx43CT to further investigate the mechanism(s) governing this regulation. Here, we utilize circular dichroism and nuclear magnetic resonance (NMR) were used to validate the TM4-Cx43CT for studying channel gating and optimize solution conditions for structural studies. The data indicate that, unlike the soluble Cx43CT, the TM4-Cx43CT is structurally responsive to changes in pH, suggesting the presence of the TM4 facilitates pH-induced structural alterations. 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).

Keywords::

• Cx43
• detergent micelles
• NMR
• TFE

Acknowledgments

This work was supported by the United States Public Health Service Grants GM072631 and HL039707, the Nebraska Research Initiative funding for the Nebraska Center for Structural Biology, and the Eppley Cancer Center Support Grant P30CA036727. (The authors regret this information was omitted from the Early Online version of the article published ahead of print on 01 06 2010.)

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.