Neurons and β-cells of the pancreas express connexin36, forming gap junction channels that exhibit strong cationic selectivity

Abstract

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(γ)s 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.

Fig. 1

Schematics of time-lapse imaging using a dual-fluorescence excitation mode. a Whole-field excitation (vertical, blue, dashed arrows) of the cell pair with two patch pipettes. Emitted light of dye (yellow) in the pipette, and cell 1 creates a large background of scattered light around the cell pair. b The same as in a but excitation light is focused only to cell 2. This eliminates excitation of dye in cell 1 and the pipette attached to it. c Molecular formulas of positively charged fluorescent dyes, EAM-1 and EAM-2

Fig. 2

Permeability of Cx36-EGFP GJ channels. a, b Images of HeLaCx36-EGFP cell pair. a Overlapped phase contrast (gray) and EGFP (green) images; shadows of two patch pipettes are accentuated by continuous lines, and the yellow arrow shows the position of the junctional plaque. b Fluorescent image of LY obtained using whole-field excitation 5 min after the patch was open to cell 1. There was no detectable LY signal in cell 2; cells were electrically coupled, g_j = 5 nS. c, d Examples of time course of changes in LY (c) and EAM-2 (d) fluorescence in cell 1 (FI₁) and cell 2 (FI₂) and in single-channel permeability (P_γ). Arrows point to the moments of patch opening in cell 1 and cell 2

Fig. 3

Voltage gating of HeLaCx36-EGFP GJs. a, b Averaged and normalized g_j – V_j plot (black solid line in b) obtained in response to V_j ramps (shown in a). White line is a fitting curve obtained using a S4SM and global optimization. c Dependencies of open probabilities of A (P_H,A) and B (P_H,B) hemichannels on V_j derived from the S4SM

Fig. 4

Permeability of Cx36– EGFP GJ channels to EtBr and DAPI. Representative images of HeLaCx36-EGFP cell pairs showing transfer of EtBr (a) and DAPI (b) recorded using whole-field excitation. Enhanced view of nucleus in cell 2 (inset in b) shows a gradient of fluorescence with higher intensity on the junctional side. Examples of time course of changes in EtBr (c) and DAPI (d) fluorescence in cell 1 (FI₁) and cell 2 (FI₂). Arrows point to the moments of patch opening in cell 1 and cell 2

Fig. 5

a Averaged single-channel permeability of homotypic Cx36–EGFP GJs for different dyes. b Single GJ channel permeabilities of different Cxs. Bars show averaged P_γs of GJ channels for AFl-350 (gray) and LY (white)