Abstract
The similar dense, protein-rich and detergent-resistant characteristics of postsynaptic densities (PSDs) and gap junctions led us to investigate the distribution of gap junctions and their constituent connexins in CNS subcellular fractions containing PSDs. Western blot analysis showed these fractions to be enriched in both neuronal and glial connexins, namely, connexin26, connexin30, connexin36 and connexin43. Connexins were retained in these fractions after treatment with n-lauroyl sarcosine to remove loosely associated proteins. Confocal double immunofluorescence confirmed the presence of connexins in PSD fractions and showed a near total co-localization of glial connexin30 and connexin43, demonstrating preservation of inter-connexin relationships that have been observed in vivo. In contrast, none of the connexins were co-localized with the PSD structural protein PSD-95, indicating their lack of direct association with PSDs. These results show that PSD preparations contain significant levels of connexin proteins, which appear to remain assembled as gap junctions. Thus, protocols used to isolate PSDs may serve as a basis for development of methods to isolate CNS gap junctions, which would aid biochemical identification of regulatory and structural proteins associated with these structures.
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