We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P γ), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P γ of Cx36 for cationic dyes (EAM-1+ and EAM-2+) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350−). In addition, P γ for Lucifer yellow (LY2−) is approximately fourfold smaller than that for AFl-350−, which suggests that the higher negativity of LY2− significantly reduces permeability. The P γ of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the P γs of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P γ of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K+, a major charge carrier in cell–cell communication.
The Journal of Membrane Biology – Springer Journals
Published: Jun 30, 2012
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