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

Abstract

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.

The Bioactive Phenolic Agents Diaryl Ether CVB2-61 and Diarylheptanoid CVB4-57 as Connexin Hemichannel Blockers

“…In order to analyse the functionality of the Cx hemichannels formed in the membranes of transfected cells, we performed dye-uptake experiments [ 11 , 34 , 35 , 36 ]. The functionality of the Cx hemichannels was recognised as an accelerated increase in fluorescence intensity of the dye ethidium bromide (Etd) in the cells in response to the removal of external Ca 2+ , as compared to control conditions with 2 mM external Ca 2+ ( Figure 2 a).…”

Unnexins: Homologs of innexin proteins in Trypanosomatidae parasites

“…We found that most parasites exhibited only a single homolog, except T. cruzi , T. brucei , and T. rangeli , which possessed five, three, and two, respectively. The expression of two or more homologs could allow the formation of heteromeric channels formed by different subunits as occurs in mammals with Cx26/Cx30 (Naulin et al, 2020), Cx26/Cx46 (Schadzek et al, 2018), Cx43/Cx46 (Hoang et al, 2010) hemichannels among others.…”

The Bioactive Phenolic Agents Diaryl Ether CVB2-61 and Diarylheptanoid CVB4-57 as Connexin Hemichannel Blocker

“…In order to analyse the functionality of the formed Cx hemichannels in the membrane of transfected cells, we performed dye uptake experiments [11], [34], [35], [36]. The functionality of Cx hemichannels was recognized as an accelerated increase of fluorescence intensity of the dye ethidium bromide (Etd) in the cells in response to the removal of external Ca 2+ as compared to control conditions with 2 mM external Ca 2+ (Figure 2a).…”

“…For this work the expression vectors pEF1a B1-hCx26-B2-IRES-GFP, pEF1a B1-hCx46-B2-IRES-GFP [34], [60] were used and constructed by gateway cloning. The gene for human connexin (hCx) isoforms hCx26 or hCx46 were cloned in the vector pEF1a which has an IRES element between the gateway cassette and the reporter GFP.…”

“…Table 1. Primers used for the BP-cloning to generate the various entry clones [34]. The transfected N2A cells or HeLa cells were fixed with an acetone/methanol mix (1:2) for 5 min at -20 °C and blocked with 1% bovine serum albumin (BSA) in phosphate buffered solution (PBS) for 30 min at 37 °C.…”