Eb. Trexler et al., The first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels, BIOPHYS J, 79(6), 2000, pp. 3036-3051
Intercellular channels formed of members of the gene family of connexins (C
xs) vary from being substantially cation selective to being anion selective
. We took advantage of the ability of Cx46 to function as an unapposed hemi
channel to examine the basis of Cx charge selectivity. Previously we showed
Cx46 hemichannels to be large pores that predominantly conduct cations and
inwardly rectify in symmetric salts, properties suggesting selectivity is
influenced by fixed negative charges located toward the extracellular end o
f the pore. Here we demonstrate that high ionic strength solutions applied
to the extracellular, but not the intracellular, side of Cx46 hemichannels
substantially reduce the ratio of cation to anion permeability. Substitutio
n of the first extracellular loop (E1) domain of Cx32, an anion-preferring
Cx, reduces conductance, converts Cx46 from cation to anion preferring, and
changes the I-V relation form inwardly to outwardly rectifying. These data
suggest that fixed negative charges influencing selectivity in Cx46 are lo
cated in E1 and are substantially reduced and/or are replaced with positive
charges from the Cx32 E1 sequence. Extending studies to Cx46 cell-cell cha
nnels, we show that they maintain a strong preference for cations, have a c
onductance nearly that expected by the series addition of hemichannels, but
lack rectification in symmetric salts. These properties are consistent wit
h preservation of the fixed charge region in E1 of hemichannels, which upon
docking, become symmetrically placed near the center of the cell-cell chan
nel pore. Furthermore, heterotypic cell-cell channels formed by pairing Cx4
6 with Cx32 or Cx43 rectify in symmetric salts in accordance with the diffe
rences in the charges we ascribed to E1. These data are consistent with cha
rged residues in E1 facing the channel lumen and playing an important role
in determining Cx channel conductance and selectivity.