Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Juan A. Orellana, Emilio Díaz, Kurt A. Schalper, Aníbal A. Vargas, Michael V.L. Bennett, Juan C. Sáez

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4′,6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.

Original languageEnglish (US)
Pages (from-to)603-609
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume409
Issue number4
DOIs
StatePublished - Jun 17 2011

    Fingerprint

Keywords

  • Affinity constants
  • Connexons
  • Hill coefficient
  • Maximal transport capacity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this