Alteration of Cx43:Cx40 expression ratio in A7r5 cells

Abstract

Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.

Zonula Occludens-1 Alters Connexin43 Gap Junction Size and Organization by Influencing Channel Accretion

“…And third, C-terminally tagged Cx43 constructs have been shown to co-oligomerize with each other and with untagged Cx43 (Lauf et al, 2001;Sarma et al, 2002). Based on knowledge of the relative expression levels of the two Cx43 species (Figure 2, A and B), and assuming random codistribution of connexins within connexons ( Figure 2C), a probability equation (Burt et al, 2001) was used to generate the expected frequency distributions of Cx43:Cx43-GFP connexon stoichiometries ( Figure 2D). The probabilistic connexon distributions suggest that when native Cx43 is expressed in HeLa Cx43-GFP cells at levels both necessary and sufficient for normalization of GJ organization (i.e., Cx43 viral expression at MOIs 5-10; Figure 1), the majority of connexons (Ͼ65%) must be composed predominately of native Cx43 (Ն4 per connexon); furthermore, nearly all connexons (Ͼ99%) must contain at least one native Cx43 ( Figure 2D).…”

Characterization of the Structure and Intermolecular Interactions between the Connexin40 and Connexin43 Carboxyl-terminal and Cytoplasmic Loop Domains

“…Cx43 and Cx40 can hetero-oligomerize, leading to the formation of heterologous gap junctions (12, 14 -16). The physiological consequences of hetero-oligomerization on gap junction intercellular communication remain unclear, although functional studies demonstrate that these structures display biophysical properties that do not correspond to their homomeric forms (14,15,(17)(18)(19)(20). Because the sensitivity of different homomeric gap junctions to biochemical modulators varies widely, the question arises as to what types of interactions occur between connexins to integrate regulatory signals in heteromeric channels.…”

Engineering Cardiac Cell JunctionsIn Vitroto Study the Intercalated Disc

“…Determination of unitary channel conductances, γ j showed not only a general shift toward higher values, but also an appearance of a bimodal distribution, with a peak at 20 -30 pS. Although the explanation for these changes, and especially the appearance of a peak at low conductance values, remains in part speculative, it may indicate emergence of a population of homomeric-homotypic Cx45, (Desplantez et al, 2011;Moreno et al, 1995;Elenes et al, 2001) Cx40 interaction (formation of Cx40/Cx43 heteromeric connexons and heterologous gap junction channels) have produced controversial results ( [Burt et al, 2001;Valiunas et al, 2001] and discussion in reference ]). While overlap of Cx43 and Cx40 immunosignals in atrium is undisputed, it remains to be shown, whether and how Cx43 and Cx40 combine in individual gap junction channels.…”