Optimizing the solution conditions to solve the structure of the connexin43 carboxyl terminus attached to the 4th transmembrane domain in detergent micelles

Rosslyn Grosely, Fabien Kieken, Paul L. Sorgen

Research output: Contribution to journalArticle

8 Scopus citations

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; S255I382) 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; G178I382]) 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 2 nd extracellular loop (G178-V196).

Original languageEnglish (US)
Pages (from-to)23-33
Number of pages11
JournalCell Communication and Adhesion
Volume17
Issue number2
DOIs
StatePublished - Apr 2010

Keywords

  • Cx43
  • Detergent micelles
  • NMR
  • TFE

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology

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