Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions

Abstract

Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding how this variation contributes to the control of intercellular signalling, both in the adult and during embryonic development, is an important problem that would be aided by reagents that interfere with gap junctional communication through specific connexins. We have begun to address this issue by raising antibodies to peptides derived from connexin43 and connexin32. Connexin43 peptides were located in the amino terminus, cytoplasmic loop and carboxytail. Connexin32 peptides came from the cytoplasmic loop and the first extracellular loop. Immunoblotting and immunostaining properties of purified IgGs were characterized on mouse heart, liver and the 8- to 16-cell mouse embryo. Effects on transfer through gap junctions were assessed in the fully compacted 8-cell mouse embryo by co-injection with Lucifer Yellow or Cascade Blue. Embryos were maintained in culture to assess the developmental consequences of injection. Peptide competition was used to confirm the specificity of immunostaining and inhibition of dye transfer. All connexin specific antibodies recognized their parent connexin on immunoblots and showed no 43/32 cross-reactivity. The connexin32 extracellular loop antibody recognized both connexin 32 and 43 on immunoblots, as predicted by the amino acid sequence homology in this region, but did not immunostain intact gap junctions. Connexin specific antibodies that immuno-stained showed the predicted connexin specificity. Antibodies to either connexin43 amino acids (AA) 1–16 (amino terminus) or AA 101–112 (cytoplasmic loop) neither immunostained nor prevented functional communication through 8-cell embryo gap junctions. Antibodies to AA 123–136 and AA 131–142 in the cytoplasmic loop immunostained heart and 8-cell embryo gap junctions and blocked transfer through them with high efficiency. Fab' fragments were equally effective. Peptide competition showed that both antibodies contained epitopes within AA 131–136 of connexin43. Antibodies against AA 313–324 in the carboxytail immunostained heart and the 8-cell embryo and, as IgGs, prevented dye transfer. Fab' fragments were ineffective. All connexin43 antibodies that blocked gap junctional communication between cells of the 8-cell mouse embryo induced non-communicating cells subsequently to withdraw from compaction.(ABSTRACT TRUNCATED AT 400 WORDS)

The Role of Connexin Hemichannels in Inflammatory Diseases

“…The amino acid sequences of Cx43 ELs have been frequently used as templates for the design of mimetic peptide blockers. Significantly, 43Gap26 (VCYDKSFPISHVR) and 43Gap27 (SRPTEKTIFII) are among the first peptide mimetics of Cx43 ELs [ 32 , 33 ]. These peptides were initially shown to inhibit gap junctional intercellular communication (GJIC) by reducing gap junction formation presumptively through their complementary interaction with the ELs, thereby preventing the docking of opposing Cx43HC [ 4 , 12 , 16 , 32 , 33 ].…”

“…Significantly, 43Gap26 (VCYDKSFPISHVR) and 43Gap27 (SRPTEKTIFII) are among the first peptide mimetics of Cx43 ELs [ 32 , 33 ]. These peptides were initially shown to inhibit gap junctional intercellular communication (GJIC) by reducing gap junction formation presumptively through their complementary interaction with the ELs, thereby preventing the docking of opposing Cx43HC [ 4 , 12 , 16 , 32 , 33 ]. The blocking effects of 43Gap26 and 43Gap27 on Cx43HC have been tested in a broad variety of cells and were shown to occur within minutes after administration, suggesting that existing CxHC are indeed targeted by these peptide blockers [ 34 , 35 ].…”

Mechanisms of Connexin Regulating Peptides

“…Connexin-mimetics initially were synthesized for use as epitopes for connexin antibody production [130,131]. The blocking antibodies raised using these peptides against the EL and IL domains of several connexins were tested as GJ blockers and demonstrated some effect in reducing dye transfer in chick embryos [116]. However, EL loop antibodies Gap7M (EL1/2), Gap11 (EL1/2 Cx32), Gap15 (IL of Cx43), and Gap17 (CT Cx40) were found to be ineffective in functional testing and were not able to reduce connexin-associated contractile responses in rabbit arterial tone [92].…”

“…Despite this, Dahl et al described that peptides, used to generate antibodies, were effective in reducing Cx32 GJ formation and signaling in oocytes [132]. In 1995, Becker et al found that connexin-mimetic peptides, mimicking the sequences of the IL domain, could effectively block GJ channels when directly injected into embryo cells [116]. In the subsequent decades, a number of connexin-mimetic peptides have been generated and tested with a multitude of proposed functions including blockade of hemichannel and GJ signaling, alterations in GJ formation, and disruption of protein localization and protein-protein interactions (Table 1).…”

“…There is very little sequence homology in the connexin-IL, unlike the -EL regions, therefore peptides are presumably isoform-specific. Early peptide studies targeting the Cx43-IL, using peptides within the L2 region, GAP 13 and GAP 15 peptides, required direct injection of the peptides into cells to produce a functional block, due to the inability of the peptide to cross the plasma membrane [116]. Since then, many studies have linked cell-penetrating moieties that permit the membrane translocation of peptides.…”