Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Abstract

solubilized connexons from co-expressing cells by centrifugation through sucrose gradients or by affinity purification using a Ni-NTA column showed no evidence of mixing of Cx26 and Cx43. These results contrast with our observations in cells co-expressing other connexins with Cx43 and suggest that Cx26 and Cx43 do not form heteromeric hemichannels. Moreover, the incorporation of Cx26 and Cx43 into oligomers and into the membrane were similarly affected by treatment of co-expressing cells with brefeldin A or nocodazole, suggesting that the lack of mixing is due to incompatibility of these connexins, not to differences in biosynthetic trafficking.

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

“…Cells were transiently or stably transfected with connexin DNA using lipofectamine (Invitrogen/GIBCO) [15]. HEK293 cells stably expressing both Cx30.2 and Cx40 were generated by sequential transfection with two different expression plasmids [15,16]. For many of the electrophysiological experiments, N2a or HeLa cells were transiently transfected with connexin DNA in pTracer-CMV2 (in which GFP is driven by the EM-7 promoter and the subcloned sequence is driven by the CMV promoter); transfected cells were identified by GFP fluorescence.…”

“…Samples were analyzed by immunoblotting. We have previously utilized this procedure to identify heteromeric interactions between connexins and demonstrated the specificity of this procedure [13,14,16,19] including a variety of "negative controls". When the supernatant from cells expressing only an untagged connexin was applied to the MicroBeads, no connexin protein was found in the eluate; all connexin protein was recovered in the "flow-through".…”

“…The potential heteromeric association of Cx30.2 with other connexins was also examined using an affinity purification strategy analogous to that previously used to study formation of heteromeric connexons between Cx43 and other connexins [13,14,16]. In each experiment, one of the two co-expressed connexins contained an HA epitope tag.…”

“…For double-labeling experiments, cells were incubated simultaneously with both rabbit anti-Cx30.2 antibodies and mouse anti-Cx43, Cx45 or Cx40 monoclonal antibodies followed by anti rabbit Cy2-and anti mouse Cy3-conjugated secondary reagents. Confocal microscopy was performed as described [16]. …”

“…Moreover, for combinations of connexins that do not form heteromers (eg. Cx43 and Cx26) the untagged connexin does not co-purify with the tagged connexin [16]. …”