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.
Highlights
Gap junction channels (GJC) are formed between adjacent cells by docking of two hemichannels
It is assumed that by forming heteromeric connexons and heterotypic gap junction channels, the cells in a tissue could achieve a rectification of gap junctions as it is observed in tissue [2,3]
The goal of the present report was to test whether concatenated connexins form heteromeric connexons and heterotypic gap junction channels with clearly determined stoichiometry (Figure 1)
Summary
Gap junction channels (GJC) are formed between adjacent cells by docking of two hemichannels. It is assumed that by forming heteromeric connexons and heterotypic gap junction channels, the cells in a tissue could achieve a rectification of gap junctions as it is observed in tissue [2,3]. Using expression systems such as the Xenopus oocytes, the formation of heterotypic gap junction channels could be unequivocally demonstrated by coupling two oocytes expressing two different connexins [4]. An asparagine residue in position 168 of Cx26 or in a homologous position in other connexins belonging to the K-N group was shown to form hydrogen bonds and was essential for the docking between hemichannels of this group [11,12,13]. The authors showed that by adapting the homologous residues, heterotypic docking between Cx26 and Cx40 connexons could be achieved
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