Connexin Type and Fluorescent Protein-fusion Tag Determine Structural Stability of Gap Junction Plaques [Neurobiology]

August 11th, 2015 by Stout, R. F., Snapp, E. L., Spray, D. C.

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel sub-components of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching (FRAP) to GJs formed from connexins fused with fluorescent protein tags. We found that connexin (Cx) 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures while Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic COOH-terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin-types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.