Enrichment of neuronal and glial connexins in the postsynaptic density subcellular fraction of rat brain

The similar dense, protein-rich and detergent-resistant characteristics of postsynaptic densities (PSDs) and gap junctions led us to investigate the d istribution of gap junctions and their constituent connexins in CNS subcell ular fractions containing PSDs. Western blot analysis showed these fraction s to be enriched in both neuronal and glial connexins. namely, connexin26, connexin30, connexin36 and connexin43. Connexins were retained in these fra ctions after treatment with n-lauroyl sarcosine to remove loosely associate d proteins. Confocal double immunofluorescence confirmed the presence of co nnexins in PSD fractions and showed a near total co-localization of glial c onnexin30 and connexin43, demonstrating preservation of inter-connexin rela tionships that have been observed in vivo. In contrast, none of the connexi ns were co-localized with the PSD structural protein PSD-95, indicating the ir lack of direct association with PSDs. These results show that PSD prepar ations contain significant levels of connexin proteins, which appear to rem ain assembled as gap junctions. Thus, protocols used to isolate PSDs may se rve as a basis for development of methods to isolate CNS gap junctions, whi ch would aid biochemical identification of regulatory and structural protei ns associated with these structures. (C) 2001 Elsevier Science B.V. All rig hts reserved.