Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32*43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32*43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32*43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32*43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter.
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