Schadzek P et al. (2018), Concatenation of Human Connexin26 (hCx26) and H...

XB-ART-55298

Int J Mol Sci 2018 Sep 13;199:. doi: 10.3390/ijms19092742.

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Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels.

Schadzek P , Hermes D , Stahl Y , Dilger N , Ngezahayo A .

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Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue.

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Species referenced: Xenopus laevis
Genes referenced: gja3 gjb2

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	Figure 1. Structural organization of connexins and gap junctions. A gap junction channel is composed of two hemichannels or connexons, which consist of six connexins. Connexins have four transmembrane domains (TM), two extracellular loops (EL), one cytoplasmatic loop (CL), as well an intracellularly localized N- and C-terminus. Homomeric connexons and homotypic gap junction channels are formed by a single connexin isoform. In the postulated heteromeric connexons and heterotypic gap junction channels, different connexin isoforms are expected. The lower right pictogram shows the constructed concatemeric connexins as heterodimeric tandem.
	Figure 2. Expression of the GFP-labeled monomeric and concatemeric connexins in HeLa cells. (A) Representative micrographs of cell pairs expressing GFP-labeled hCx46, hCx26, hCx46-hCx46, hCx26-hCx26, hCx46-hCx26, and hCx26-hCx46 are shown. The cells were imaged 24 h after transfection using a confocal laser scanning microscope. The nuclei (blue) were stained with Hoechst 33342. Gap junction plaques are indicated by arrows. Gap junction plaques were found in HeLa cells expressing hCx46, hCx26, and the four different tandems. In cells expressing the tandems, a trend to accumulate the proteins in intracellular organelles was observed. (B) Quantification of the gap junction plaque area formed by the monomers and the four different tandems in HeLa cells. The plaque area was calculated using the particle analyzer of ImageJ and normalized to the number of transfected cell pairs. At least three transfections were performed per construct. The results are given as average plaque area per cell pair [µm2]. Error bars represent the SEM. The data were evaluated by a one-way ANOVA and a post-hoc Tukey test ( p ≤ 0.01, * p ≤ 0.001) in comparison to hCx46 and hCx26 (### p ≤ 0.001). (C) Quantification of the relative protein amount in HeLa cells expressing the monomeric hCx46-GFP or the homodimeric hCx46-hCx46-GFP. An anti-Cx46 antibody was used for the western blotting. For the quantification, four independent replicates were analyzed by using the gel analyzer tool of the FiJi software [32]. The data was normalized to the intensity of the hCx46 monomer.
	Figure 3. Analyzing the gap junction functionality by dye transfer experiments. A whole-cell patch-clamp configuration with a pipette solution containing 1 mg/mL Lucifer yellow was established on a HeLa cell pair expressing GFP-labelled monomeric hCx26 or hCx46 or one of the concatemeric variants. Mock transfected cells were used as control. The first row of the micrographs shows the phase contrast images of example experiments. In the second row, the GFP fluorescence signal before a dye transfer experiment is shown. The third and fourth rows show the fluorescence signal of the tracer dye 5 min and 10 min after establishment of the whole cell configuration. For the sake of clarity, the image in the fourth row was taken after removal of the dye filled capillary. Likewise, the experiments were performed with N2A cells. The cells were transfected with IRES-GFP constructs resulting in the expression of untagged constructs in the membrane and GFP in the cytosol. As control, cells expressing only GFP were used. The experiments were performed with a pipette solution containing 1 mg/mL AMCA, which could easily be distinguished from GFP under the fluorescence microscope. The cells were considered as coupled if the fluorescence intensity, which was measured in the unpatched cell of a cell pair after 10 min, was at least twice as bright as the background, which was measured at the beginning of the experiment. The probability of coupling (bar diagrams) was estimated as ratio of the sum of coupled cell pairs per the sum of tested pairs. The results are given as average. Error bars represent the SEM. The data were evaluated by a one-way ANOVA and post-hoc Tukey test (* p ≤ 0.05, p ≤ 0.01, * p ≤ 0.01) in comparison to the control cells.
	Figure 4. Analysis of single hemichannels formed by concatemeric connexins. The stripped membrane of Xenopus oocytes, which were injected with hCx46, hCx26, or the four different concatemeric constructs, as well as the AS38 (control) cRNA 24 h before, was used to perform the inside-out patch-clamp recordings. The measurements were performed in presence of Cs+ and in absence of Cl− on both sides of the membrane. (A) Examples of single channel currents elicited by a depolarizing voltage pulse of +50 mV in absence of Ca2+ in the bath solution are shown. (B) The open probability of all measured single channels was analyzed. The error bars represent the SEM. The data were evaluated by a one-way ANOVA followed by a Tukey test (* p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001, ns: not significant). The statistical comparison showed that the presence of Ca2+ or carbenoxolone (CBX) in the bath solution significantly reduced the open probability of all tested variants.
	Figure 5. Dye uptake through hemichannels using ethidium bromide. HeLa cells were grown on coverslips to a confluency of about 40–50% and transfected with IRES-GFP-plasmids. The GFP allowed the identification of transfected cells by fluorescent microscopy. (A) Time course of dye uptake experiments by cells expressing the different variants when perfused with bath solutions containing 2 mM Ca2+, no Ca2+, and no Ca2+ but 1 mM La3+. The fine lines show the SEM spread for all measured points. The symbols indicate the average for data points measured every 1 min. The solid lines indicate the part of the curves that was used to estimate the dye uptake rate. (B) Quantification of the dye uptake rate (Etd AU/min) for all tested variants and the backbone control in absence or presence of Ca2+ or La3+. The error bars represent the SEM. The data were evaluated by a one-way ANOVA and a post-hoc Tukey test (* p ≤ 0.05, *** p ≤ 0.001, ns: not significant).
	Figure 6. Recordings of dual whole-cell patch-clamp experiments. N2A cells were cultured and transfected with the different IRES-GFP-plasmids. As control, cells expressing only soluble GFP in the cytosol were used. Twenty-four hours post transfection, the dual whole-cell patch-clamp experiments were performed. The resting membrane potential was set to −40 mV for both cells. One cell of a cell pair was alternatingly stepped from −120 mV to +60 mV, while the junctional currents were recorded in the other cell. The junctional currents (ΔI2) recorded during the 250 ms-long voltage pulses at different transjunctional potentials are shown above the current responses. Magnification of the Vj +70 mV traces of the hCx46 monomer, as well as of the hCx46-hCx46 homodimer, showed several simultaneously open channels, with a large conductance of ~193 pS (13.5 pA step), and low conductance of ~72 pS (5.05 pA step) and ~121 pS (8.49 pA step). Similar steps were observed for Cx46 by other authors [38,39,40,41]. The control cells showed only the background noise, which was below 2 pA (grey band), indicating that the fluctuations of about 5 pA were conducting substates.

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