TY - JOUR
T1 - Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species
AU - Orellana, Juan A.
AU - Díaz, Emilio
AU - Schalper, Kurt A.
AU - Vargas, Aníbal A.
AU - Bennett, Michael V.L.
AU - Sáez, Juan C.
N1 - Funding Information:
This work was partially supported by CONICYT (24080055 to JAO), FONDECYT (1111033 to JCS), FONDEF (D07I1086 to JCS and KSC), NHI (NS55363 to MVL) and Anillo (ACT71 to JCS) grants.
PY - 2011/6/17
Y1 - 2011/6/17
N2 - 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.
AB - 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.
KW - Affinity constants
KW - Connexons
KW - Hill coefficient
KW - Maximal transport capacity
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U2 - 10.1016/j.bbrc.2011.05.031
DO - 10.1016/j.bbrc.2011.05.031
M3 - Article
C2 - 21600880
AN - SCOPUS:79959219603
SN - 0006-291X
VL - 409
SP - 603
EP - 609
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -