The First Extracellular Loop Domain Is a Major Determinant of Charge Selectivity in Connexin46 Channels

Abstract

Intercellular channels formed of members of the gene family of connexins (Cxs) vary from being substantially cation selective to being anion selective. We took advantage of the ability of Cx46 to function as an unopposed hemichannel 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 of 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. Substitution 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 located in E1 and are substantially reduced and/or are replaced with positive charges from the Cx32 E1 sequence. Extending studies to Cx46 cell-cell channels, we show that they maintain a strong preference for cations, have a conductance nearly that expected by the series addition of hemichannels, but lack rectification in symmetric salts. These properties are consistent with preservation of the fixed charge region in E1 of hemichannels, which upon docking, become symmetrically placed near the center of the cell-cell channel pore. Furthermore, heterotypic cell-cell channels formed by pairing Cx46 with Cx32 or Cx43 rectify in symmetric salts in accordance with the differences in the charges we ascribed to E1. These data are consistent with charged residues in E1 facing the channel lumen and playing an important role in determining Cx channel conductance and selectivity.

New roles for astrocytes: Gap junction hemichannels have something to communicate

“…Most simply, the extracellular region of the hemichannel when it is at the substate conductance could have a very different configuration to that of the same region in the cell-cell channel. V j or fast gating to the substate in cell-cell channels is thought to involve the cytoplasmic end of the channel, and charges implicated in the voltage sensor are located there; however, other charged residues affecting gating are found at the beginning of the first extracellular loop [45,50].…”

“…An open hemichannel could well exhibit asymmetry in fixed charges that causes rectification, whereas the asymmetry is neutralized in the cell-cell channel [45,51].…”

“…Most simply, the extracellular region of the hemichannel when it is at the substate conductance could have a very different configuration to that of the same region in the cell-cell channel. V j or fast gating to the substate in cell-cell channels is thought to involve the cytoplasmic end of the channel, and charges implicated in the voltage sensor are located there; however, other charged residues affecting gating are found at the beginning of the first extracellular loop [45,50].Although not yet adequately characterized, there are differences in singlechannel rectification in hemichannels. An open hemichannel could well exhibit asymmetry in fixed charges that causes rectification, whereas the asymmetry is neutralized in the cell-cell channel [45,51].…”

“…V j or fast gating to the substate in cell-cell channels is thought to involve the cytoplasmic end of the channel, and charges implicated in the voltage sensor are located there; however, other charged residues affecting gating are found at the beginning of the first extracellular loop [45,50].…”

Gap junction channel structure in the early 21st century: facts and fantasies

“…Other domain swap studies showed that the charge selectivity of connexin pores can be controlled by E1 (Cx46, Cx32; [40]), suggesting that E1 contributes to the pore wall. Point mutations in the NT produced changes in the single channel current-voltage relations consistent with electrostatic effects on the permeating ions (Cx32; [32]).…”